Pulsed field electrophoresis chambers, accessories and method of utilization for separation of DNA molecules

ABSTRACT

Methods of use, accessories and chambers, optimal for performing Pulsed Field Gel Electrophoresis (PFGE) of DNA molecules in ‘Contour Clamped Homogeneous Electric Field’ (CHEF) and ‘Transversal Alternating Field Electrophoresis’ (TAFE) systems, are provided herein. DNA molecules are rapidly separated in the minigels of these chambers. The sizes of chambers and accessories are determined by the separation between the opposite polarity electrodes; which is comprised between 6.2 and 15 cm. Reproducibility of molecule separation is achieved because the accessories warrant homogeneous electric resistance in the buffer and minigels. Chambers allow a high-throughput sample format using the reagents efficiently. It is attained excluding the non-useful electrophoresis zones For a better optimization, TAFE chambers have several useful electrophoresis zones (UEZ), each carrying a minigel. One or more UEZ can be activated at will in the electrophoresis, to vary the number of minigels, the number of samples and the amount of buffer among the experiments. TAFE chambers having ‘inverted electrode configuration’ with the cathodes at their bottom are presented.

CLAIMS OF PRIORITY

This application claims priority to Cuban Application Nos. CU 0135/00filed Jun. 7, 2000, CU 0306/00, filed Dec. 27, 2000 and PCT/CU01/00003,filed Jun. 7, 2001.

REFERENCE TO RELATED APPLICATION

The present invention is related to the field of Molecular Biology andmore particularly, it refers to Pulsed Field Gel Electrophoresischambers of the ‘Contour Clamped Homogeneous Electric Field’ (CHEF) and‘Transversal Alternating Field Electrophoresis’ (TAFE) systems. Thisinvention is also related to the use of these chambers for theseparation of DNA molecules and a method for the selection of theconditions of electrophoresis.

BACKGROUND OF THE INVENTION

Pulsed field gel electrophoresis (PFGE) dates from 1984, when SchwartzD. C and Cantor C. (Cell, 37, 67–75, 1984; U.S. Pat. No. 4,473,452)observed that applying electric pulses that periodically alternatedtheir direction in a certain angle in relation to an agarose gel, largeintact DNA molecules were resolved as band patterns. The authors alsodetermined that the separations of the molecules essentially depended onthe duration of the electric pulses. Later, it was determined that thegeometry of the lines of force of the approximates alternatingelectrical fields, the strength of them, the experimental temperature,the ionic strength of the buffer solution and the concentration of theagarose gel were important factors that influenced the resolution thatcould be achieved among DNA molecules. (Birren B. and Lai E. AcademicPress. New York, 1993, p 107, 111, 129, 131, 135; López-Cánovas L. etal., J. of Chromatogr. A, 1998, 806, 123–139; López-Cánovas L. et al.,J. of Chromatogr. A, 1998, 806, 187–197).

Pulsed field electrophoresis renders the separation of the DNA moleculesas band patterns Each pattern is formed in the lanes of the separationgels after the electrophoresis. Altogether, the agarose plugs containingthe immobilized DNA molecules are loaded into each well of the gel,then, the molecules migrate along the length of the gel and form theband patterns during the electrophoresis. That is why this type ofelectrophoresis has associated a method for the preparation of intactDNA molecules immobilized in plugs of gel. These molecules can be ornot, digested with restriction endonucleases before the electrophoresis.

Several systems to perform PFGE have been developed. They arecharacterized for having chambers in which the electrodes are placed indifferent arrangements Among these chambers are the OFAGE (OrthogonalField Alternating Gel Electrophoresis, Carle C. F. and Olson M. V.Nucleic Acids Res. 1984, 12, 5647–5664) CHEF (‘Contour ClampedHomogeneous Electric Field’, Chu G. Science 234, 1986, 1582–1585), TAFE(‘Transversal Alternating Field Electrophoresis’, U.S. Pat. No.4,740,283), FIGE (‘Field Inversion Gel Electrophoresis’, U.S. Pat. No.4,737,251 of Carle G. F. and Olson M. V) arrangement of electrodes, andthe minichambers MiniTAFE and MiniCHEF (Riverón, A. M. et al., Anal.Lett, 1995, 28, 1973–1991; European Patent Application EP 0 745 844).

All these systems are characterized by having electronic circuitry foralternating the electric fields and accessories for the preparation ofthe gel. There are also accessories for the preparation of the samples.The systems differ among them by the complexity of the electronics toenergize the electrodes and to switch the orientation of the electricfield They also differ by their capacity to render straight paths ofmigration of the band patterns The possibility to obtain straight pathsof migration is essential when the comparison of the patterns of severalsamples is wished, while the simplicity of the electronics facilitatesand makes cheaper the construction of the systems. Among the systemsmentioned, only three render straight paths of migration of themolecules:

-   -   1.—the CHEF system, which has electrodes that are clamped to        predetermined electrical potentials, electrodes that are        arranged in a hexagonal contour around a submarine gel that is        horizontally positioned;    -   2.—the TAFE system, in which the electrophoresis is performed in        submarine gels that are positioned vertically in the chamber and        uses fields transversal to the surfaces of the gel; and    -   3.—the FIGE system, in which the electrophoresis is performed in        horizontal submarine gels that are positioned in conventional        electrophoresis chambers, which have two electrodes. In this        system, the orientation of the electric field is reverted        periodically These systems have in common that in their chambers        the gel is symmetrically crossed by the lines of force of the        electric fields that are generated at the electrodes with        opposite polarity in the electrode arrangement. In that gel, the        samples containing the intact DNA molecules are loaded. In all        these chambers exist zones where the force lines of the electric        field do not act on the molecules. The zone of the chamber that        contains the gel and is crossed by the lines of force of the        electric field that directly interact with the molecules will be        denominated here as useful electrophoresis zone (UEZ) Whereas        the zones of the chamber crossed by the lines of force of the        electric field that do no act on the molecules will be        denominated here as non-useful electrophoresis zones (NEZ) All        existing chambers to perform PFGE have a single UEZ and several        NEZ regions

The chamber and the electronics of the FIGE system are simple. FIGEchambers that allow the simultaneous analysis of many samples exist (upto 96 samples, using two combs of 48 teeth in the chamber OnePhorAllSubmarine Gel System of Jordan Scientific, BDH Catalogue BDH, 1997,Section E p 4–371), but in these chambers inversion of the mobility ofthe molecules occur (Carle G. F., Frank M. and Olson M. V. Science, vol.232, p 65–68, 1986). Due to the absence of a theory that predicts theinversion mobility in FIGE under any experimental conditions, suchinversion limits the use of these chambers to analyze the size of DNAmolecules separated and to compare their band patterns. For instance,this phenomenon will cause that two DNA molecules of different sizesmigrate the same distance in the gel, preventing their identification,excepting by means of hybridization with DNA probes. Up to now, the twoonly ways to estimate the size of large DNA molecules separated inexperiments of PFGE are

-   -   1) To compare the distances migrated by the molecules under        study to the distances migrated by the size markers and    -   2) To use equations that describes the distances migrated by the        molecules under different electrophoresis conditions and later        replace in the equations the migrated distances and the        experimental variables.

In FIGE the size markers can also suffer mobility inversion and, asmentioned above, there is no theory capable to predict the moment andconditions of appearance of such inversion. These are serious drawbacksof FIGE chambers, especially to compare many samples, for instance, inmolecular epidemiology studies. Because of these reasons the systemsmost frequently used to compare band patterns of many samples are CHEFand TAFE.

Gardiner K. et al. in their paper published in Somatic Cell Mol. Genet.1986, 12, 185–195 proposed the TAFE system. They called it “VerticalPulsed Field Electrophoresis” (VPFE) and developed an equipment whichwas disclosed in U.S. Pat. No. 4,740,283 dated Apr. 26 of 1988. Thissystem for the separation of DNA molecules uses a vertical gel of10×7.6×0.6 cm (height×width×depth) and has the electrodes arranged inparallel to the faces of the gel and across the chamber. In the chamber,each member of a pair of electrodes with opposite polarity is positionedin front of a face of the gel. The cathode is positioned at the top andnear the origin of migration and the anode far from it, at the bottom.Such electrode arrangement generates equipotential lines spanning thelength of the gel and a gradient potential or electric field, where thelines of force of such electric field cross the gel transversally. Then,along the height of the gel a gradient of electric field strength and ofthe angle formed between the lines of force of the two pair ofelectrodes are obtained. That is the reason why the molecules arecompelled to migrate during each pulse through the thickness of the gel.The resultant migration occurs in vertical direction, downward. Despiteof the existence of these gradients, all the gel points situatedwidthwise and at the same height, in relation to the plane that containsboth cathodes or both anodes, are at a same value of electric potential(equipotential lines) Thus, molecules of the same size migrate similardistances during the electrophoresis in all the lanes of the gel andmigrate following straight paths up to the same height in the gel,independently on the wells in which the samples were loaded.

Based on these principles, Beckman Instrument, Inc. (Beckman, TheGeneline System Instruction Manual, ed. Spinco Division of BeckmanInstruments, 1988), constructed the equipment called “Geneline I”, or“Transverse Alternating Field Electrophoresis System” known as TAFE.This system uses a gel of 11×7.2×0.6 cm (height×width×thickness), whichis placed between the pairs with opposite electrodes that are separated20 cm. Later, Beckman Instruments, Inc. developed the equipment“Geneline II” in which the gel was enlarged to 14.2×15×0.3 cm. TheGeneline II equipment is no longer been produced.

To resolve large DNA molecules in a band pattern, a long time isrequired in the TAFE equipments Geneline I and Geneline II. Forinstance, Geneline I needs 24 hours to render a pattern of 11 bandscorresponding to the chromosomes of the yeast Saccharomyces cerevisiae(molecules less than 1.6 Mb in length. 1 Mb=10⁶ base pairs). Thisequipment may heed up to 90 hours to separate the DNA molecules of theamoebic genome in 17 bands (Orozco E. et al., Molec. Biochem. Parasitol.vol. 59, p 29–40, 1993) TAFE chambers require a large volume buffersolution to cover the electrodes (approximately 3500 ml in Geneline II)and through the electrophoresis buffer the flow of electric current ishigh and the generated heat might be large. If in the TAFE equipment,the potential difference applied across the electrodes with oppositepolarity is increased, the maximum current output of the power supplymay be achieved. That is why, the companies recommend 10 V/cm as themaximum value of electric field (for power supplies with a maximumcurrent output of 0.4 Amp). Large heat generation in the electrophoresisimpedes the reduction of the duration of electrophoresis by increasingthe electric field strength. It has been stated that the use of elevatedvoltages or high temperatures broaden and make diffuse the bands of theelectrophoresis pattern, rendering poorly resolved bands. The advantageof the Geneline II is to permit the simultaneous analysis of 40 samples,which facilitates the comparative analysis of the electrophoresispatterns given by many samples.

Gilbert Chu (Science 1986, 234, 16, 1582–1585) developed CHEF system onthe following basis: a homogeneous electric field is theoreticallygenerated by two infinite electrodes placed in parallel at certaindistance. To simulate a homogeneous field using finite electrodes,another group of electrodes is placed in a plane, along a closedpolygon, that might be a square or a hexagon.

The x axis (y=0) of the plane is set to coincide with a side of thepolygon and zero volt is applied. The opposite side is placed at adistance ‘A’ (y=A) from the origin of ordinates and it is polarized to apotential ‘Vo’. The rest of electrodes are polarized according toV(y)=Vo·y/A. In this way, the potential generated in the interior of thepolygon is equal to those that should be generated by two infiniteparallel electrodes separated a distance ‘A’ The reorientation angleobtained by electronic permutation of the polarity between two pairs ofdifferent sides will be 90° for the square and 60° or 120° for thehexagon A method to clamp the desired potentials across the CHEFelectrodes is to set a series of resistors wired to form a voltagedivisor between potentials V(0)=0 and V(A)=Vo From each of the nodes,formed by the union of two resistors, the voltage for the polarizationof one electrode is withdrawn.

Based in these principles, the Bio-Rad Company developed the equipmentsCHEF-DR II, CHEF-DR III and CHEF Mapper (U.S. Pat. No. 4,878,008, U.S.Pat. No. 5,084,157 and U.S. Pat. No. 5,549,796). The last is the mostadvanced system. To clamp the voltages across the electrodes of thehexagonal array, the voltage divisor is wired to a transistorized systemand operational amplifiers. This electronic design warrants that thevoltages that are applied across the electrodes of the hexagonal arraywill be always correct.

The dimensions of the CHEF Mapper electrophoresis chamber are11.4×44.2×50.3 cm (height×width×depth), it weights 10.2 Kg and uses 2.2liters of buffer solution. This system uses a gel of 14×13 cm (width andlength) that is concentrically positioned with the hexagonal arrangementof 24 electrodes, whose parallel sides are separated 30 cm or more. CHEFMapper is also capable to use a wider gel where up to 40 samples can beloaded into.

The TAFE and CHEF equipments are able to separate chromosomal sized DNAmolecules. Nevertheless, a common disadvantage of the CHEF and TAFEequipments is that the chambers are unnecessarily large, because theirdimensions have not been optimized yet, particularly when thin sampleplugs are used. It has been demonstrated that the thickness of theagarose plugs that contain the DNA samples influences the resolution ofthe bands, the electrophoresis time and the length of the gel to be used(López-Cánovas L. et al. J Chromatogr. A, 1998, 806, 187–197). In thatwork, it was demonstrated that if it is wished to obtain a resolution‘X’ between two any molecules, this value is obtained in less space andless time if the bands are thinner, which is achieved if the plugs arealso thinner. Among the consequences of using large electrophoresischambers are:

-   -   I) When high electric fields are applied, the use of power        supplies with large maximum output is required. These chambers        have more than 20 cm of distance between the electrodes with        opposite polarity; therefore, the maximal electric field that        can be applied in these equipments is approximately 10 V/cm.    -   II) The experiments are long in these chambers. Two factors        influence long run duration, very low electric fields are used        (usually 6 V/cm), and samples are around 0.1 cm thickness. For        instance, normal experiments take 24 hours to obtain the        electrophoresis patterns of the eleven chromosomal bands,        corresponding to DNA molecules of Saccharomyces cerevisiae less        than 1.6 megabases (10⁶ base pairs), and up to 90 hours to        separate the 17 bands of DNA molecules from the genome of        Entamoeba histolytica (Orozco E et al, Mol. Biochem. Parasitol.        1993, 59, 29–40).    -   III) The equipments are not economical, because large amount of        expensive reagents (such as Tris and agarose) and biological        samples are used. The latter might impede certain applications        (for instance in clinical diagnosis).    -   IV) A large quantity of heat is generated in the electrophoresis        chamber when the driving force of the electrophoresis or        electric field (which depends on the applied voltage across the        electrodes and on the current intensity that flows through the        buffer solution) is increased. If the electric field is        increased (aimed to increasing the velocity of separation), it        should be done by increasing the voltage applied across the        electrodes, and therefore the current intensity By Joule effect,        the generation of heat in the chamber will increase. An        excessive increase of heat evolved will broaden and make the        bands diffuse and will provoke distortion of the electrophoresis        pattern and even entrapment of DNA molecules in the pores of the        gel and the complete absence of migration.

Nevertheless, the large volume of buffer solution filling these chambershas the advantage that the turbulences generated in the solution duringits circulation are attenuated. Altogether, the gel is so distant fromthe electrodes that any local change in conductivity near the electrodesproduced by electrolysis is diluted and will not have deleterious effectdue to the large volume of solution.

In 1995 were disclosed the MiniCHEF and MiniTAFE equipments, in whichPulsed Field Gel Electrophoresis of 8 samples loaded into a gel areperformed (Riverón A. M. et al., Anal. Lett, 1995, 28, 1973–1991;European Patent Application EP 0 745 844) These equipments overcame thedeficiencies of the above-mentioned systems. The MiniCHEF as well as theMiniTAFE use thin samples thinner than 0.1 cm and they allow theapplication of higher electric fields rendering adequate resolutionsamong the bands of the patterns. Therefore, in the miniequipments, thechromosomes of the yeast Saccharomyces cerevisiae were resolved in 4 to5 hours.

The separation between the opposite electrodes of minichambers is small,thus allowing the construction of smaller chambers and the use of lessvolume of buffer to cover the electrodes and the gel (Riverón A. M. etal., Anal. Lett, 1995, 28, 1973–1991; European Patent Application EP 0745 844, Bull. 1996/49). That is why in MiniCHEF and MiniTAFE, lowamount of heat is not evolved, even if high electric fields are appliedThe samples loaded into the gels of these equipments need a small amountbiological material (Riverón A. M. et al., Anal. Lett, 1995, 28,1973–1991). Furthermore, they save laboratory bench space.

The authors of these equipments demonstrated the feasibility ofperforming PFGE in gels that are not long. For instance, gels of 4 cm inlength were used. By means of the use of mini-equipments López-CánovasL. et al. (López-Cánovas L. et al., J Chromatogr A, 1998, 806, 187–197)demonstrated that plugs thicker than 0.1 cm render thick bands and themolecules need more time and more gel length to be separated. Inaddition, the use of thick samples does not improve the quality of theelectrophoresis pattern and does not reveal more bands.

The mini-equipments proposed by Riverón A. M. et al. to perform PulsedField Gel Electrophoresis have chambers whose sizes are calculated basedon the existence of other equipments of larger dimensions (Riverón A. M.et al., Anal. Lett, 1995, 28, 1973–1991; European Patent Application EP0 745 844). Therefore, they can inherit errors of the equipments fromwhich they were designed. In fact, the mini-equipments inherited fromthe large chambers an open system for the preparation of the gel and theabsence of a proper system for attenuating turbulences of the bufferflowing throughout the chamber In the patent application and the relatedpapers, the effects of the reduction of the volumes of the buffer andthe gel on the electrophoresis pattern are not mentioned That is, thequestion whether this volume of buffer is enough to attenuate theturbulences during its circulation, or whether the irregularities in thegel and the differences in dimensions of the plugs influence the qualityof the bands patterns that are obtained, are not resolved yet Thesetroubles increase with miniaturization, because miniaturizationmagnifies the manufacture errors. For instance, if a meniscus of 0.1 cmheight is formed in a gel of 1 cm of thickness, the error in the heightof the gel would be 10%, while that same error in a 0 4 cm thick gelrepresents 25%. Therefore, the magnification of the errors byminiaturizing the system can become critical factors to obtainreproducible bands patterns

The relevant parameter of pulsed field electrophoresis equipments is theseparation between the electrodes, because it determines the values ofelectric field that can be applied. It also determines the driving forceof the molecules, the dimensions of the chambers, the systems thatshould be used to homogenize the variables of the electrophoresis, thelength of the separation gel, the thickness of the plugs where thesamples are included and the width of each sample

If the separation between the electrodes with opposite polarity is notoptimal, for instance, if it is too large, then the dimensions of thegel, the chamber and the number of samples that can be applied in thosegels will not be optimal. If the plugs do not have the proper thicknessand size, an excessive quantity of gel will be used and largeelectrophoresis time will be consumed. In addition, the shape anddistribution of the dimensions of the chambers as well as the existenceof a single UEZ region determines that the reagents consumed in thesechambers will not be used optimally. Therefore, the desired goal is todevelop chambers with optimal dimensions, which allow the application ofhigh electric fields; chambers whose internal dimensions vary accordingto the number of samples they analyze and that the electrophoresis runtime to be short without loosing resolution or high capacity of sampleanalysis.

From the above reasons, it is concluded that.

-   -   Large chambers of the current PFGE systems are not optimal,        because the separation between electrodes with opposite polarity        is unnecessary large and the same amount of reagents is used,        independently on the number of samples to be studied.    -   The chamber dimensions are not optimal. The dimensions of the        chambers (height, width and depth) do not warrant that the        current flowing through the chamber does not exceed easily the        output limits of the power supplies for PFGE, and thus, and do        not separate the molecules fast at high electric fields.    -   In order to increase the number of UEZ, relevant constructive        modifications have to be carried out in the existing chambers.        They may affect the proper functioning of the systems. This        factor influences the optimization of the use of reagents.

As it was already mentioned, the TAFE chambers (Geneline I, Geneline II)and MiniTAFE have an electrode platform to accommodate a gel (or twogels in Geneline II) Electrode platform whose width is equal to thewidth of the chamber and its height depends on the separation betweenthe electrodes with opposite polarity (that is, they have an UEZregion). In the gel(s), so samples can be applied as many as is allowedby its width, the width of the samples and the separation between them.The equipments that have an UEZ region use a constant volume of buffersolution to cover its electrodes.

If the number of samples that is desired to be simultaneously analyzedexceed the maximal capacity of analysis of the UEZ of any of thementioned chambers (for instance, more than 8 in MiniTAFE, more than 20in Geneline I and more than 40 in Geneline II), it would be necessary toperform several electrophoresis. Therefore, the comparison of theresulting band patterns will not be reliable. For instance, when it isdesired to characterize the genome of 100 isolates of a particularmicroorganism, either from a collection of isolates of thebiotechnological industry, or infected human, animal or vegetables Then,these three chambers have insufficiencies in their capacity tosimultaneously analyze more than 8, 20 or 40 samples, respectively, orare insufficient the possibilities to increase the capacity of analysis.Therefore, when it is necessary to perform co-electrophoresis of manysamples to compare the band patterns of the DNA molecules of the samplesthe maximal capacity analysis of TAFE (Geneline I, Geneline II) andMiniTAFE can be exceeded.

A known solution, that would increase twofold the capacity of sampleanalysis of the mentioned chambers, is the implemented in the FIGEchamber OnePhorAll. This consists in positioning two combs in the gel ofthe UEZ, one of them at the beginning of the gel and the other in themiddle of it. However, in the TAFE system, the samples loaded into thewells formed by the two combs would not be subjected to the sameelectric field nor to identical reorientation angle; thus molecules ofsimilar size would migrate different distances in the gel and the bandspatterns would not be comparable.

Another possible solution could be to construct wider chambers withwider gels and UEZ zones. This solution was implemented in Geneline IIand supposedly; it should allow the analysis of many samples (more than40). That is why Geneline II was designed as a non-deep but wide andtall chamber. However, it was necessary to place dielectrics between theelectrodes and the gel in order to obtain the characteristic anglegradient of TAFE system. These dielectrics considerable slow down theelectrophoresis runs. On the other side, the electric current flowingthrough the chamber depends directly on the cross section area thatencounters the ionic flux. Thus, through these very tall and widechambers flow elevated current, exceeding those flowing in Geneline Iand the former VPFE. Therefore, by applying low voltages, the maximumcurrent (Imax), or power (Pmax) outputs of the power supply, are reachedin less time. For instance, Macrodrive I, LKB: Imax=0.4 Amp, Vmax=500volts, Pmax=200 Watts; PowerPack 3000, Bio-Rad, Cat. 1998–1999: Imax=0.4Amp, Vmax=3000 volts, Pmax=400 Watts; Consort E802, Cat. BDH 1997:Imax=2 Amp, Vmax=300 volts, Pmax=300 Watts (Vmax, Imax and Pmax maximumvoltage, current and power outputs, respectively). Then, in this type ofchambers is impossible to increase the electric field strength to reducethe electrophoresis time Low electric fields unnecessary enlarge theduration of PFGE experiments, fact that reduces the spectrum ofapplications of these chambers in the fields of the science andtechnology that require the rapid obtainment of results. In addition,Geneline II uses larger volume of reagents than current chambers. Infact, Beckman Instruments has discontinued Geneline II.

Wider miniTAFE chambers could be designed (maximum applicable electricfield 25 V/cm for approximately 6 cm in width), because the chambers areneither deep nor tall The cross section area of miniTAFE could beincreased if the electric current (I) flowing through the buffer at agiven ‘E’ value (for instance 8–10 V/cm) does not exceed the maximumoutput of the existing power supplies. These chambers use less volume ofbuffer solution than the current TAFE, Geneline I and Geneline IIchambers. In miniTAFE, the band patterns would be obtained in a relativeshort time However, such broad UEZ would need a very wide minigel, whichwould present difficulties in its casting and handling. Additionally,several minigels could be accommodated, but according to formula I (seeforward), this chamber would not be efficient when analyzing a smallnumber of samples. When it is necessary to analyze a small number ofsamples, for instance 8, the analyzing capacity of the gels of the TAFEGeneline I and Geneline II is largely wasted, because they have a singleUEZ that can accommodate 20 or 40 samples, respectively The reagentsused in PFGE experiments are expensive. The equipments would efficientlyuse their capacities of separation of DNA molecules if the volumes ofreagents used each time in the chambers would depend on the number ofsamples to be analyzed. This is impossible in chambers of a single UEZbecause they use a constant volume of reagents. The volume of reagentsin excess (ER %) that is used in chambers of a single UEZ can be definedasER(%)=100.0·(Nt−N)/Nt  (I)where:

-   -   Nt: Maximal number of samples that can be loaded in a minigel    -   N: Number of samples really analyzed in an experiment    -   (Nt−N): Number of samples not loaded in the gel

The ER values of the Geneline II and MiniTAFE systems are shown in Table1 When few samples are analyzed, ER increases in both chambers,evidencing that they use reagents in excess when few samples areapplied. Although miniTAFE (data in column 2 Table 1) uses less volumeof reagents than TAFE, this volume neither varies with the number ofsamples analyzed. Hence, the volume of reagents used by the TAFEGeneline I, Geneline II and MiniTAFE chambers is constant andindependent on the number of samples to be analyzed, fact that impedesits optimal use.

In addition, the buffer solution becomes exhausted duringelectrophoresis. That is why, to make an optimal design of the shape anddimensions of the chambers, it is necessary to know the time that thebuffer solution takes to exhaust.

The chambers of the TAFE system to separate DNA molecules use avertically placed gel and its cathodes are located at the top of thechamber. Then, the direction of migration is parallel to the vector ofthe gravity force. To avoid accidents with the electrodes while placingthe gel in the chamber, Geneline I have two removable electrodeplatforms and the gel is accommodated into the chamber before suchplatforms are placed.

TABLE 1 Excess of reagents (ER %) used in Geneline II and MiniTAFE N(No. of TAFE GL-II MiniTAFE samples Bc = 3500 Bc = 325 loaded) ER (%) ER(%) 1 97.5 87.5 2 95.0 75.0 3 92.5 62.5 4 90.0 50.0 5 87.5 37.5 6 85.525.0 7 82.5 12.5 8 80.0 0.0 9 77.5 — 10 75.0 — 11 72.5 — 12 70.0 — 1367.5 — 14 65.0 — 27 32.5 — 40 0.0 — —: Means that the gel does not havethose wells. ER: Percentage of reagents used in excess. GL-II: GenelineII. Bc: Total volume of buffer solution filling the chamber, in ml.

To implement this solution it is necessary to place the electrodes andthe gel in relation to the platforms in the proper position. However,this double positioning of the electrodes in the platforms and theplatforms with respect to the gel can vary the relative dispositionbetween the gel and the electrodes. Therefore, this aspect should beimproved in the design of the chambers.

As it was mentioned, in the existing chambers there are zones crossed bylines of force of the electric field that do not act directly upon themolecules loaded into the gel (non useful electrophoresis zone, NEZ).These regions do not play an essential role in the separation of the DNAmolecules.

The miniequipments previously reported are not optimum, because they donot have any system to attenuate turbulences of the buffer solutionflowing through the chamber neither to prepare gel withoutirregularities nor to form thin sample plugs of similar sizes andshapes.

Up to now, the attention has been focused on maintaining constant thevoltage across the electrodes of the chambers during theelectrophoresis. It is particularly notorious in the CHEF chambers, inwhich is necessary to set a given voltage value in each electrode of thehexagonal array. However, the quality of the band patterns and theexperimental reproducibility are affected by variations in the voltageand other factors The reproducibility of the band patterns is alsoaffected by the factors that provoke non-homogeneity of the currentflowing through the buffer filling the chambers and the factors thatcould distort the lines of force of the electric field.

These other factors have not been completely considered in PFGE systemsFor this reason, the current systems can give as results distorted bandpatterns. These problems are relevant in the miniequipments forelectrophoresis. They are:

-   -   The chambers do not have simple devices to attenuate the        turbulences of the buffer flowing through the chamber and the        external heat exchange.    -   The accessories to cast the gels do not avoid the formation of        irregularities and defects in the electrophoresis gel.    -   The accessories to immobilize the DNA molecules in the agarose        plugs do not warrant the formation of sample plugs with        dimensions similar to those of the wells of the agarose gel.        There are not devices to achieve a good alignment of the sample        plugs in the migration origin either.    -   There are not devices to warrant maintaining the electrodes        stretched.

The mentioned aspects affect the obtainment of straight and reproducibleband patterns in the different lanes of the gel. These aspects alsoaffect the band pattern reproducibility during different electrophoresisruns in the same or in several equipments.

On the other hand, the chambers for pulsed field gel electrophoresis arefilled with a buffer solution that is circulated between the chamber andan external heat exchanger From the potential applied across theelectrodes is generated the electric field or driving force of themolecules and the buffer solution is the medium through which theelectric field is established. The physicochemical processes occurringin the buffer during the electrophoresis, as the electrolysis, thebuffer heating by Joule-effect and the variations of the concentrationof the ions of the buffer provoke non-homogeneities in the conductingproperties of the buffer solution. The temperature, concentration andothers variables affect the viscosity of the buffer, and thus theelectric field generated through the buffer as well as the movement ofthe DNA molecules Thus, DNA migration is affected in different fashionsthroughout the chamber, when any of these variables are randomly changedThe electrolysis also affects the buffer conductivity. The buffer in thechamber is constantly exchanged with thermostated buffer at constanttemperature. It is accomplished by using a peristaltic pump. Therefore,it is intended to maintain homogeneous and constant the properties ofthe buffer solution. The buffer flow velocity should warrant the totalexchange of the chamber's buffer in a few minutes. However, at a givenbuffer injection velocity, turbulences in the chambers are generated.Then, local non-homogeneity of the applied electric field is generated,which affects the movement of the DNA molecules

The resulting band patterns depend on the variations of the conductivityin the buffer solution of the chamber and the presence of turbulences inthat buffer. The turbulences are increased if the buffer is circulatedat high flow velocities. The turbulence, vortices or waves locallychange the height of the buffer, modifying randomly and regionally theelectric resistance values of the buffer in the chamber. The variationsin the electric current flowing through the different regions of thechamber modify the DNA migration and generate distorted DNA bandpatterns.

The equipment CHEF MAPPER from Bio-Rad considers this problem (CHEFMapper XA Pulsed Field Electrophoresis System. Instruction Manual andApplication Guide p 4. Bio-Rad). The Bio-Rad CHEF has two small chambersbelow the main chamber floor at the front and rear of the main chamber.They are used for buffer circulation and priming the pump. Buffer entersthe main chamber through six holes in the floor near the top. A flowbaffle just in front of these holes prevents gel movement. However, thissystem is not efficient to attenuate buffer turbulence, mainly when highflow velocity is used

Neither the TAFE nor the miniequipments have any system for attenuatingthe turbulences of the buffer flowing throughout the chamber, which is adisadvantage. It is easy to sense that the turbulences are more harmfulin the miniequipments that use less amount of buffer. For example, theturbulences in the CHEF Mapper chamber, filled with 2.2 L, areattenuated easier than in the miniCHEF and miniTAFE that use ten timesless buffer

As it was mentioned, the PFGE large chambers attenuate in certain extentthe height of buffer oscillations. However, the miniequipments for PFGEare relatively recent. maybe for this reason the development of a systemfor attenuating the turbulences of the buffer flowing through thesechambers has not been a focus of attention.

The gels used in the CHEF and TAFE equipments of large dimensions aswell as in the mini-equipments matches with the mold where they arecast, a comb is inserted and the molten agarose is poured. While theagarose is solidifying, the mold is not covered. Then, because of thesurface tension of the molten agarose, it wets the walls of the moldforming a meniscus The meniscus is formed between the wells of the gelor in the walls of the gel mold. The mold to prepare the TAFE gel has alid, but it has not accessories to avoid the meniscus formation amongthe teeth of the comb. The accessories to pour the gels of the miniCHEFand miniTAFE do not have cover, and then the meniscuses are formed inthe sites above-mentioned.

The gel is the medium through which occurs the migration of DNAmolecules during the electrophoresis. The presence of meniscuses at theedges of the gel or between the wells of the gel modifies the electricresistance in the gel and consequently the electric current The regionalchanges of the electric current flowing through the gel affect the DNAmigration in such regions. These changes are essential if meniscuses areformed The gel wells are the origin of migration of the molecules;consequently, if the irregularities in these zones provoke changes inthe velocity of migration of the molecules, the migration boundarieswill be distorted. Then, these distortions will be maintained during theelectrophoresis process, finally giving distorted patterns in the gellanes Any gel irregularity in any other region will also affect themolecule migration through such region From the point of view of theband pattern reproducibility, the accessories to prepare the gel and themethod to use it are important. The designs of efficient systems forpulsed field gel electrophoresis has been focused in afford chamberswith different electrode configurations and an electronic circuitrysuitable to switch the electric field and impose the voltages. Theproperties of the accessories to prepare the gels have not beenexhaustively considered

As it was mentioned, the pulsed field gel electrophoresis includes themethodology for the preparation of intact DNA molecules immobilized ingel plugs. To do it, it is necessary to have molds to form the sampleplugs.

The existing molds are the following:

-   -   A mold to form similar and single plugs (Cantor C. R. and        Schwartz D. C., U.S. Pat. No. 4,473,452);    -   A mold to forms long and flat ribbons that are cut to provide        single plugs;    -   A mold to form long agarose rods that are cut to provide single        plugs (Birren B. and Lai E. Pulsed Field Gel Electrophoresis: A        Practical Guide, Academic Press, New York, 1993, 29–30).

Usually, the above molds generate sample plugs of dimensions larger thanthe wells of the gel. For this reason, it is recommended to cut theplugs with the aid of a blade or other instrument (CHEF Mapper XA PulsedField Electrophoresis System. Instruction Manual and Application Guide p40, Section 7. Catalog Numbers 170–3670 to 170–3673. Bio-Rad).

In the current chambers (CHEF, TAFE, miniCHEF and miniTAFE), theinequalities of the sample plugs provoked by cutting them after theirpreparation, affect the quality of the electrophoresis patterns. It isknown that the sample plug thickness has influence upon the DNAresolution and the electrophoresis time. However, the effect of theshape and dimension inequalities of the sample plugs upon theelectrophoresis patterns has not been thoroughly studied. The effectsprovoked by a bad alignment of the sample plugs lengthwise the origin ofmigration have not been studied yet. The researchers have used thesample plugs makers mentioned in the above paragraph. However, thesemolds do not include devices to cut the sample plugs with identicalshape and dimensions and matching with the gel wells.

If it is considered that the band patterns obtained in each gel lane atthe end of the electrophoresis depend on the fact that molecules ofsimilar sizes are moving together from the wells toward the bottom ofthe gel, the importance of the accessories to prepare the sample plugsand align them in the gel wells will be understood. That is, themigration boundary should move forming a thin and straight band. Whenthe migration boundary is deformed in the origin of migration, it willbe maintained deformed during the electrophoresis, because in thechamber does not exist any device or force to correct the movement. Theflaws preparing the sample plugs and troubles in their alignment in thegel wells are exactly reproduced in the bands separated in the patterns,and might provoke tilted and undulated bands.

In the U.S. Pat. No. 5,457,050 of 1995 of G H Mazurek, was disclosed amold and a processing chamber to perform the cell immobilization andtreat the cells inside the mold. Depending on the material used toconstruct such mold, it could be disposable or reusable. Besides thatsample plug preparation could be longer using this processing chamber,said mold does not have associated a device to cut the sample plugs and,thus, plugs of similar sizes are not warranted to be obtained.

On other hand, the equipments TAFE Geneline I and Geneline II fix itsfour platinum electrodes between two parallel acrylic sheets (Beckman,The Geneline System Instruction Manual, ed. Spinco Division of BeckmanInstruments, 1988). One of the end of each electrode goes toward the lidof the chamber, outside of the useful electrophoresis zone. It is donewith the aim of connecting the platinum wire to a plug in the lid of thechamber. In this way, it is warranted the electrical continuity betweenthe circuitry and buffer solution as well as the polarization of theelectrodes. The platinum wire in the lid is insulated with a plasticcapillary with high dielectric constant. As it is known, the platinumelectrodes become thinner during pulsed field gel electrophoresis andduring their use, the electrodes slacken and become bent and undulatedfor several zones Them, the system used in the TAFE to set theelectrodes has the disadvantage that to pull tight the electrode theexperimenter must dismount the electrode from the lid and this is verydifficult.

When the electrodes become bent, undulated or slacken, the equipotentiallines in the gel and the force lines of the electric field become alsodistorted provoking that bands do not migrate in a sharp and straightboundary.

By the other side, the way to fix the electrodes in the TAFE equipmentswastes a portion of platinum wire. For example, the Geneline I usesapproximately one meter of platinum wire, while the active electrodesrequire only thirty centimeters. The Geneline II has a similar design.

In the CHEF Mapper, the electrodes (J-shape) are fixed on supports madeof material with high dielectric constant, in such a way that one of itsends passes through the support (CHEF Mapper XA Pulsed FieldElectrophoresis System. Instruction Manual and Application Guide p 4 and65, Section 7. Catalog Numbers 170-3670 to 170-3673. Bio-Rad). Thesupports are inserted into the floor of the chamber. In this way, theplatinum wire passes through the floor of the chamber and is connectedto the circuitry to clamp the voltage across the electrodes. To seal thefloor of the chamber a silicone sealant and rubber O-rings pressed downwith a nut are used. The fixing of electrodes in the CHEF Bio-Rad savesplatinum wire because the electrodes are not so large and they do nothave to pass out of the buffer. However, it is not warranted that theseelectrodes are maintained stretched and consequently slight deformationsof the lines of force of the electric field can occur.

The disclosed MiniTAFE and MiniCHEF equipments (Riverón A. M. et al.,Anal. Lett, 1995, vol. 28, 1973–1991; European Patent Application EP 0745 844, Bull. 1996/49) have extended the electrode platinum wire abovethe buffer solution level in the chamber as the TAFE equipment does. Inthis way, it is warranted the necessary communication between theelectrodes and the electronic circuitry to polarize them. The regions ofthe platinum wire that do not act as electrode are insulated with tubingmade of material with high dielectric constant to avoid the contact ofsuch platinum wire with the buffer The TAFE chamber uses electrodes thatare at least as long as the width of the gel and are suspended betweenthe sidewalls of the chambers. During the use, the electrodes slackenand undulated, so the band patterns can be distorted. Besides, itrepresents an additional expense of platinum wire and them the chambersare more expensive.

MiniTAFE equipments separate the S. cerevisiae chromosomes at highelectric fields (22 V/cm), giving a suitable resolution between thebands of the electrophoresis patterns in the minigels (Riverón et al.,Analytical Letters, vol. 28, p 1973–1991, 1995). Besides it, using theminiTAFE the S. cerevisiae chromosomes can be resolved in 5 hours, at 8Volt/cm and 20° C. Small separation between opposite electrodes permitsthe construction of small chambers and the use of small buffer volume tocover the electrodes (350 ml) When across the miniTAFE electrodes agiven voltage is applied, that is, a certain value of electric fieldstrength ‘E’ is applied, then the heat dissipation is less than thoseobtained in TAFE equipments if the same ‘E’ would be applied. Thesamples plugs loaded into the minigels of the mini-equipments need smallamount of biological material and the plug thickness ranges from 0.1 to0.05 cm. They reduce the electrophoresis time and contribute to givesharp bands in the patterns (López-Cánovas et al., J Chromatography A,806, p 187–197, 1998). In the minigels can be loaded as many sampleplugs as permitted by the minigel width. For example, for a gel of4.0×4.0×0.5 cm (width, height and depth) can be loaded up to 10 sampleplugs of 2.5 mm in width and spaced apart 1 mm

Despite of the mentioned advantages, the refereed equipments haveinadequacies that limit their application in the analysis of numeroussamples. In particular, when the number of samples to be analyzedchanges considerably among the experiments. Several of theseinadequacies are related to the shape and arrangements of the chamberdimensions as well as the existence of a single UEZ.

There are methods to select the run conditions in the PFGE equipments.For example, the CHEF Mapper from Bio-Rad has both auto-algorithm andinteractive algorithm options (CHEF Mapper XA Pulsed FieldElectrophoresis System. Instruction Manual and Application Guide. 31-40Catalog Numbers 170-3670 to 170-3673. Bio-Rad). Both options permit tocalculate the pulse time, the duration of the ramps of pulse time,reorientation angle, electric field and the optimum electrophoresis timeto separate the DNA molecules of a given sample In contrast to theauto-algorithm, that assumes constant values for the variables, theinteractive algorithm permits to change the time, temperature andconcentration of the buffer and the type and concentration of agaroseBoth algorithms make the calculations based on empiric and theoreticaldata, collected during five years of experiences (Bio-Rad Catalogue.Life Science Research Products 1998/99. Bio-Rad Laboratories, 185).However, the manufacturers recommend entering to the auto-algorithm DNAsizes lower and larger than the limits of the range to be optimized.They also recommend to be considered that both algorithms can giveerroneous results such as DNA mobility inversion in the mid-range of thegel, when extremely wide size ranges are entered in both algorithms.

There are other empirical expressions giving the pulse time that wouldseparate a group of molecules which sizes are between a given size andother superior one called RSL (Resolution Size Limit) (Smith D. R.Methods 1, 1990, 195–203). However, this relation is only valid in someexperimental conditions and does not predict the resolution between anypair of molecules. There is also a function calculating the approximateconditions of the electric field and pulse time that separate a givenset of molecules (Gunderson K. and Chu G. Mol. Cell. Biol., 1991, 11,3348–3354). It should be noted, that such function only permits toestimate the approximate values of these two variables, but does notgive the migration of the molecules at any experimental condition.

Despite many theoretical studies about the reorientation of the DNAmolecules during PFGE have been performed (Noolandi J., Adv.Electrophoresis, 1992, 5, 1–57; Maule J., Mol. Biotech., 1998, 9,107–126), they have not given practical results, useful in thelaboratory It means that they do not generated methods that permit theeasy selection of the experimental conditions that separate a givengroup of molecules.

The equations proposed by López-Cánovas L. et al (López-Cánovas L. etal., J. Chromatogr. A, 1998, 806, 123–139) to describe DNA migration inPFGE have not been extended to select the experimental conditionsapplicable in any equipment when the pulse time, electric field andtemperature varies.

Up to now, the experimental conditions applicable in PFGE equipments arethe result of the experience of the PFGE's user, more than the resultsof equations describing DNA migration in PFGE. There is not a securemethod to predict the pulse and run times hat should be applied at anyconditions. That method is particularly important when the minichambersof the mini-equipments are used, because in the miniequipments can beused high electric field strength. The use of such high electric fieldstrength is not frequent in the rest of the PFGE systems.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to chambers for pulsed field gelelectrophoresis of the ‘Contour Clamped Homogeneous Electric Field’(CHEF) or ‘Transversal Alternating Field Electrophoresis’ (TAFE)systems, the accessories and the methods for their use

The chambers of this invention are used to separate large DNA moleculesby pulsed field gel electrophoresis (PFGE), done in miniequipments andminigels, as well as in chambers using multiple minigels. Chambers,accessories and methods herein disclosed have applications for typingstrain collections of the food industry, as well as strain collectionsof scientific research and clinical microbiology laboratories. They alsofind applications in molecular epidemiological studies and in thedetermination of sources of contamination in the biotechnologicalindustry. The chambers can be used to type multi-drug-resistantbacteria, to characterize the genome of mammalian and vegetable speciesas well as to study human hereditary diseases. In this last application,new rapid and reproducible diagnosis methods can be developed.

The present invention provides pulsed field gel electrophoresis chambersof the CHEF and TAFE systems. They have optimal dimensions and permit toapply high electric fields.

They also permit to perform co-electrophoresis of many or few samples inmultiple minigels as well as to reduce the running time durationmaintaining the resolution among the molecules and the high-throughputcapacity of analysis.

In the invention, it is assumed the existence of systems to energize theelectrodes with the proper voltage values that should be set in CHEF andTAFE chambers. A system, as the one reported by Maule (Maule J. andGreen D. K. Anal. Biochem. 1990 191, 390–395) or other similar, issuitable for the proper electrode polarization. It is also assumed thatare available a power supply, an external heat exchanger, a circulatorto thermostat the buffer solution in the chamber, as well as thechemical and biological reagents to carry out the electrophoresisprocess of the large DNA molecules.

The invention, disclosed herein, provides:

Minichambers for pulsed field gel electrophoresis (PFGE) in the CHEF andTAFE systems, having a single useful electrophoresis zone (UEZ), fromwhich the non-useful electrophoresis zones (NEZ) have been eliminated.The minichambers circulate the buffer solution at high flow velocity butavoiding turbulences in the buffer flowing throughout the chamber. Theyalso permit the fast separation of the molecules in band patterns thatare reproduced in all lanes of gels, band patterns that are alsoreproduced among electrophoresis performed at different moments.

TAFE multiminichambers, having separations between the oppositeelectrode pairs as those of minichambers, two or several UEZ thataccommodate a minigel each one, and chambers from which non-usefulelectrophoresis zones were eliminated. These chambers have ahigh-throughput capacity of sample analysis but they can analyze fewsamples maintaining its optimization and rapidity of analysis. It isforeseen that the reagent consumed in these chambers depends on thequantity ‘N’ of samples that is going to be analyzed in each experiment.It is also foreseen that the user can vary the number of UEZ that willbe used in each experiment and the shortening of the electrophoresisrunning time.

Accessory sets to attenuate turbulence of then buffer flowing throughoutthe chamber, to prepare minigels of flat surfaces, with the absence ofirregularities, as well as to cast sample plugs of homogeneous shapesand sizes similar to those of the gel wells into which they will beloaded.

Methods for using the pulsed field gel electrophoresis chambers, methodsthat include a method to calculate the electrophoresis run time at anyvalues of electric field and temperature used in the chambers.

The chambers, accessories and methods of this invention, permit the fastseparation of large DNA molecules in minigels, using agarose in aconcentration range between 0.5 and 1.5%. In particular, the chambers,accessories and methods provided herein have the following distinctivecharacteristics:

They use of rectangular or square shaped minigels into which can beloaded up to 200 sample plugs. The number of samples depends on theminigel width, which in turn, depends on the separation between theelectrodes with opposite polarity in CHEF chambers and on the width inthe TAFE chambers. The volume of buffer solution also depends on theseparation between electrodes with opposite polarity and the width ofthe chamber.

The chambers provide reproducible results because the chambers as wellas the accessories warrant homogeneous electric current throughout thebuffer solution and good alignment of the sample plugs in the migrationorigin. They also warrant stretched electrodes.

The chambers are able to separate rapidly, at least in 2.5 hours, theDNA molecules of sizes up to 2 megabase pairs.

The chambers are provided with a method to calculate the electrophoresisrun time, if the electric field, temperature and pulse time applied inthe electrophoresis process are provided

A.—The Calculation of the Minigel Dimensions, the Area of the Chambersand the Number of Samples That Can be Loaded into the Wells.

To perform these calculations, we will name ‘d’ to the separationbetween the pairs of electrodes with opposite polarity The dimensions,recommended for the width and length (in cm) of the CHEF minigels, aswell as the area of the floor of the chamber without NEZ regions andhaving a single UEZ with a hexagonal array of electrodes, are given by

$\begin{matrix}{{{width}\mspace{20mu}{of}\mspace{20mu}{the}\mspace{20mu}{rectangular}\mspace{20mu}{minigel}} = {d\text{/}\left( {2 \cdot {\cos\left( {30{^\circ}} \right)}} \right)}} \\{= {d\text{/}1.732}} \\{{length} = {d \cdot \left\{ {1 - {1{\text{/}\left\lbrack {2 \cdot {\cos^{2}\left( {30{^\circ}} \right)}} \right\rbrack}}} \right\}}} \\{= {d\text{/}3}} \\{{{side}\mspace{20mu}{of}\mspace{20mu}{the}\mspace{20mu}{square}\mspace{20mu}{minigel}} = {d \cdot \left\{ {1 - {1{\text{/}\left\lbrack {2 \cdot {\cos^{2}\left( {30{^\circ}} \right)}} \right\rbrack}}} \right\}}} \\{= {d\text{/}3}} \\{{{area}\mspace{20mu}{of}\mspace{20mu}{the}\mspace{20mu}{chamber}\mspace{20mu}{floor}} = {\left\lbrack {2 + \left( {d\text{/}{\cos\left( {30{^\circ}} \right)}} \right)} \right\rbrack \cdot \left\lbrack {6 + d} \right\rbrack}} \\{= {\left\lbrack {2 + \left( {d\text{/}0.87} \right)} \right\rbrack \cdot \left\lbrack {6 + d} \right\rbrack}}\end{matrix}\quad$

NEZ regions were eliminated from the chamber, because they do not playan essential role in the separation of DNA molecules. In addition, thechamber side walls should be separated 1 cm from the electrodes, and thesystem for attenuating turbulences of the buffer flowing throughout thechamber flow (explained further) should occupy 2 cm in the buffer inletand outlet regions. These considerations explain the constant values of2 and 6 in the formulas of the area of the chamber floor If ‘d’ isbetween 6.2 and 15 cm, then

-   -   the width ‘a’ of rectangular-shaped minigels is comprised        between 3.6 and 8 7 cm.    -   the length is between 2.1 and 5 cm,    -   the area is between 7.6 and 43.5 cm²,    -   the side of square/shaped minigels is comprised between 2.1 and        5 cm,    -   the area is between 4.4 and 25 cm²,    -   the area of the floor of the chamber is between 111.3 and 404.1        cm²

The buffer level in the chamber must surpass 0.3 cm the minigel height,thus the amount of buffer volume is defined by:buffer volume={[2+(d/cos(30°))]·[6+d]}·(0.3+minigel thickness)

If the minigels thickness is between 0.35 and 0.5 cm, then the amount ofbuffer volume will be between 72.3 and 323.3 ml.

The maximum number of plugs that can be loaded in minigels is definedaccording to their width ‘a’ (in cm) as:number of plugs to be loaded in the minigels=(a−0.2)/0.25

Where, for rectangular-shaped minigels of 3.6 and 8.7 cm, the maximumnumber is comprised between 13 and 34 plugs, respectively.

The length (in cm) of TAFE minigel in minichambers that have a singleUEZ or several UEZ (multiminichambers) is:

$\begin{matrix}{{{length}\mspace{20mu}{of}\mspace{20mu}{the}\mspace{20mu}{minigel}} = {d \cdot {\sin\left( {31{^\circ}} \right)}}} \\{= {d \cdot 0.515}}\end{matrix}\quad$and the minigel width ‘a’ is the width of the UEZ. When the TAFEchambers have a single UEZ, the minigel width coincides with the chamberwidth; whereas, if the chambers have larger number of UEZ, themultiminichambers will have two or several minigels, being their widthsequal to the width of each UEZ.

The area (in cm²) of the walls supporting the minigel and the electrodesis given byarea=[2+1.4·d]·[2+0.54·d]−1.02·[1+0.54·d] ²

If ‘d’ is between 6.2 and 15 cm, the minigel length will be between 3.2and 77 cm, whereas the area of each wall, supporting the minigel and theelectrodes, will be between 37.8 and 147.8 cm².

In TAFE chambers, the less is the distance ‘d’ between the pairs withopposite electrodes, the less is the cross section area, and, thus,higher electric fields could be applied without significant incrementsof the initial electric current ‘I_(o)’ and the power in the chamberTherefore, to construct TAFE chambers having large width ‘L’ (chamberwidth or dimension parallel to electrodes), it is convenient that thedistance ‘d’ be small This warrants that ‘E’ values separating fast thechromosomes without ‘I_(o)’ surpassing the maximum output of theconventional PFGE power supplies may be applied.

Although the chambers have small separations between oppositeelectrodes. optimal chambers are only those that are wide enough tosupport simultaneously several minigels, which can be excluded (or not)from the experiments at will as well as the corresponding buffer volume.The subdivision of a very wide minigel in several less wide minigels isattained efficiently if the chamber is subdivided in several UEZ. If allsamples that the minigels admit are loaded into them, then the chamberswill be functioning at full capacity, analyzing simultaneously manysamples However, if few samples would be analyzed. its capability ofanalysis would not be put in good use and the reagents would be wastedTo avoid it, only the required UEZ must be activated, and the non-usedUEZ should be excluded. In that way, the volume of reagents used in eachexperiment will depend on the number of samples to be analyzed and,therefore, on the number of UEZ activated.

The maximum number of samples that can be simultaneously analyzed in theTAFE minigels depends on the size and number of UEZ, which in turndepends on the maximum width (‘L’) of the chamber. The chamber widthdetermines the electric current withdrawn from the power supply. Thence,the limiting factors for constructing wide TAFE chambers are theelectric current and power outputs of the power supplies available.Thus, the knowledge of the power supply characteristics permits tocalculate ‘a priori’ the maximum width that TAFE chambers could have, ifequations describing the electric current circulating through thechamber are available.

To obtain the equations describing the electric current circulatingthrough the chamber, a TAFE chamber was constructed. It has a distancebetween the pairs with opposite electrodes in the range defined for TAFEminichambers and 316 mm width (‘L’). NEZ were eliminated from thischamber, and accessories were designed to permit to vary the innerdimensions of the chamber from 7 cm to the width ‘L’ of the chamber,thus, allowing to have ‘n’ TAFE chambers of different widths. To obtainthe equations, the specific conductance ‘p’ (mho.cm⁻¹) of 0.5×TBE buffer(1×TBE: 89 mM Tris, 89 mM boric acid and 2 mM EDTA, pH 8.3) is fitted toa function of the Tris molar concentration and the experimentaltemperature ‘T’ (° C.):ρ=5.190·10⁻³·[Tris]^(0.8461) ·e ^(0.02214) ·T  (II)

Here, the initial electric current ‘I_(o)’ (in Amperes) flowing throughthe chamber was taken as depending on the electrolyte resistance, whichis given by the relation between the vessel constant ‘Cv’ (in cm⁻¹) andthe specific conductance ‘ρ’. The vessel constant depends on theseparation between electrodes with opposite polarity and on the crosssection area ‘A’ (cm²) that the chamber presents to the flow of theelectric current.

The chambers do not have regular geometric shapes due to the eliminationof the NEZ; therefore, the vessel constants (Cv=d/A) need to bedetermined for different widths and shapes. To determine them, thefollowing procedure was developed:

-   -   The cell constant ‘Cv(cond)’ of a conductivity meter is        determined.    -   The chamber is filled with a solution of any given electrolyte,        which is maintained at fixed temperature.    -   The conductivity of said electrolyte is measured using the        calibrated cell of the conductivity meter ‘G(cond)’.    -   The conductivity of said electrolyte is measured by connecting        the electrodes of the electrophoresis chamber to the measuring        plugs of the conductivity meter ‘G(chamber)’.

It is easy to deduce that ‘Cv(chamber)’ is:Cv(chamber)=[G(cond)·Cv(cond)]/G(chamber)  (III)

For each of the ‘n’ TAFE chambers having different width ‘L₁’ (where ‘i’is between 1 and ‘n’) ‘Cv(chamber)_(i)’ is determined. Then, thefunction relating ‘Cv’ and ‘L’ can be obtainedCv(chamber)₁ =f(L ₁)  (IV)

Hence, in chambers of ‘L₁’ width, the electrical resistances ‘Re_(i)’,given by electrolytes of ‘ρ_(j)’ conductivity, are given by:Re _(i) =Cv(chamber)₁/ρ_(j)  (V)

Since the initial studies on conductivity, it is known that if a voltageis applied using a direct current power supply, the measured electriccurrent flowing through the electrolyte does not depend on ‘Re’ alone.In fact, polarization of the electrolyte occurs, thus, reducing thevalues of the electric field and the circulating electric current. Forthis reason, to design TAFE chambers of variable width, functionsdescribing the attenuation of the electric field due to electrolytepolarization must be available.

The electric field attenuation in the electrophoresis chambers ofdifferent widths and geometric shapes can be estimated if ‘Re’ is known(equations II, IV y V) and if it is considered that the resistance (R)measured in the buffer solution filling the chamber can be modeled astwo resistances in series, ‘Re’ and ‘Rp’R=Re+Rp,  (VI)where ‘Rp’ plays the role of an additional resistance induced byelectrolyte polarization Then, according to Ohm's lawV _(DC) =I _(DC) ·R  (VII)where V_(DC) is the voltage applied using the direct current powersupply and I_(DC) is the direct current measured: If increasing valuesof V_(DC) are applied and I_(DC) values are measured, then ‘Rt’ valuescan be calculated and ‘Rp’ estimated from the knowledge of ‘Re’ values(equation V) in that chamber. In that way, the function relating ‘Rp’ to‘Re’ and to ‘V_(DC)’ can be obtainedRp=f(Re, V _(DC))  (VIII)

By using the above equations, it is possible to predict the initialcurrents (‘I₀’) that would be withdrawn applying V_(DC) voltages tochambers having different widths and filled with buffer solutions ofdifferent conductivities and setting the electrophoresis at varioustemperatures. Therefore, the maximum width that each chamber could havecan be estimated for each existing power supply of known electriccurrent, voltage and power output. The best chamber width is the one,which withdraws electric current ‘I₀’ and power ‘P’ that do not surpassthe maximum output values of the power supply. The voltage to get that‘I₀’ or that ‘P’ is the maximum voltage that can be applied in saidchamber

To design TAFE multiminichambers, the prediction of ‘I₀’, is not enough,it is also necessary to consider the buffer exhaustion. That is, theincrements of the electric current circulating through the buffer of thechamber, due to buffer exhaustion should not surpass the electriccurrent and electric power limits of the power supply during theelectrophoresis. For that reason, it is necessary to develop equationsdescribing buffer exhaustion as the electrophoresis time elapses. Thetime needed to exhaust the buffer should depend, at least, on ‘E’. Theequation describing the buffer exhaustion constant (‘k’, in Ohm. t⁻¹)can be obtained assuming that, if a voltage ‘V_(DC)’ (in volts) isapplied across the electrodes, the electric current ‘I_(DC)t’ (inAmperes) flowing through the chamber at any electrophoresis moment isgiven byI _(DC)(t)=V _(DC) /R(t)  (IX)where,R(t)=R+kt  (X)k=f(E)  (XI)and ‘R’ is calculated according to the equation VI. Thus, by the use ofthese equations. ‘I₀’ can be predicted, as well as the variations ofelectric current flow during the electrophoresis., This calculationallows knowing the moment of replacing the buffer solution during eachexperiment. These equations can be obtained empirically, adding buffersolution into the chamber, regulating the temperature, applying thevoltage across the electrodes and finally monitoring the electriccurrent (‘It’) flowing through the buffer during the time (‘t’).Further, regression methods are used for describing the relationshipamong the variables.

The relationships II to XI can be used to calculate the maximum width‘L’ of the TAFE multiminichamber, which depends on the distance ‘d’between electrodes, the conductivity ‘ρ’ and the buffer temperature ‘T’.The width also depends on the applied electric field, which isrestricted by the maximum electric current ‘Imax’ and power ‘Pmax’outputs of the power supply used to energize the multiminichamber. Thatis,L=f(Imax, d, ρ, T, Emax)  (XII)L=f(Pmax, d, ρ, T, Emax)  (XIII)

The maximum width ‘L’ of TAFE multiminichambers is the smaller of thetwo ‘L’ values obtained using the functions XII and XIII.

Based on the equations XII and XIII and using power supplies that giveup to 2 amperes and 300 watts of electric current and electric poweroutputs, respectively, the widths ‘L’ of TAFE multiminichambers canreach up to 50 cm. In said chambers, the distance between pairs withopposite electrodes can reach up to 15 cm, and 0.5×TBE buffer can beused at 30° C. as maximum temperature These multiminichambers can besubdivided in UEZ If all ZUE are used in the electrophoresis, theelectric fields can be increased up to 8 V/cm, whereas if several UEZare inactivated, the electric field can be reached up to 25 V/cm. Thenumber of UEZ can vary between 1 and 30.

The area of the minigel of TAFE chambers that have one or multiple UEZ(multiminichambers) is:minigel area=d·sin(31°)·L/UEZ _(total)=d·0.515·L/UEZ _(total)the volume of the buffer solution added to the chamber depends on thewidth of the selected chamber, and it is calculated in the followingway:buffer volume=[(2+1.4·d)·(2+0.54·d)−1.02·(1+0.54·d)² ]·L·UEZ _(act) /UEZ_(total)where,

UEZ_(act): number of UEZ activated during the electrophoresis,

UEZ_(total): number of UEZ in the chamber.

The subdivision of TAFE chambers in UEZ improves the efficiency of saidchambers It is made evident taking again the formula I and defining:

Bc: Volume of reagents in ml (of the buffer solution or the agarose)that requires the chamber. Bzue: Volume of reagents in ml required byeach UEZ, being each UEZ used to separate a maximum of ‘NM’ samples.Bnt: Volume of reagents in ml required when a number ‘Nzue’ of UEZ areactivated. Bnt = Nzue · Bzue. Nt: Maximum number of samples that can beloaded in all UEZ that were activated Nt = NM · Nzue. (Nt − N) Number ofsamples that were not loaded in the experiment. (Bnt/Nt): Volume ofreagents in ml required by each sample. (Nt − N) · (Bnt/Nt): Excess ofreagent volume when ‘N’ samples are loaded but ‘Nt” samples could beloaded.

By relating the excess of reagent volume to the reagent volume ‘Bc’needed by the chamber, and calling the result ‘ER’, ‘ER’ is:ER(%)=100.0·Bnt·(Nt−N)/(Nt·Bc)  (XIV)

By applying the relation XIV to a chamber that has four UEZ, each UEZrequiring 325 ml of buffer solution and admitting a minigel supporting13 samples, the values shown in Table 2 will be obtained for ‘ER’. Inthe example, ‘ER’ ranges from a maximum value of 23 1% if ‘N’ is 1, 14,27 or 40 samples, to a minimum of 0% if ‘N’ is 13, 26, 39 or 52 samples(Table 2). Notice that, if a single UEZ is used, the remaining threeones are inactivated and occluded, so Bnt=325 ml and Nt=13. Similarly,if the first and second miniplatforms are used only, Bnt=650 ml andNt=26, and so on. Chambers having a single UEZ are the Geneline II,Nt=40, Bc=Bnt=3500 ml and the MiniTAFE Nt=8, Bc=Bnt=350 ml. Thus, Bc=Bntand ER=(Nt−N)/Nt (Table 1). Therefore, the chambers must be subdividedin several UEZ, by subdividing or not the electrode platform, butmaintaining the capacity to perform co-electrophoresis to the desirednumber of samples, and being energized with a single power supply.Additionally, they do not waste excess of reagents. In these chambers,the volume of reagents (‘Bnt’ in ml) used during each electrophoresisdepends on the maximum number of samples to be analyzed (‘Nt’) in eachexperiment All UEZ of a TAFE chamber must be activated simultaneouslyusing a single power supply and a single system to alternate theelectric fields.

TABLE 2 Excess of reagent volume (‘ER’) in a chamber of four UEZ thatperforms transversal alternating field electrophoresis (MultiMiniTAFE)in four minigels Bc = 1300 ml ZUE1 ZUE2 ZUE3 ZUE4 Nt = 13 Nt = 26, Nt =39, Nt = 52, Bnt = 325 Bnt = 650 Bnt = 975 Bnt = 1300 N = 1 . . . 13 N =14 . . . 26 N = 27 . . . 39 N = 40 52 N ER (%) N ER (%) N ER (%) N ER(%) 1 23.1 14 23.1 27 23.1 40 23.1 2 21.2 15 21.2 28 21.2 41 21.2 3 19.216 19.2 29 19.2 42 19.2 4 17.3 17 17.3 30 17.3 43 17.3 5 15.4 18 15.4 3115.4 44 15.4 6 13.5 19 13.5 32 13.5 45 13.5 7 11.5 20 11.5 33 11.5 4611.5 8 9.6 21 9.6 34 9.6 47 9.6 9 7.7 22 7.7 35 7.7 48 7.7 10 5.8 23 5.836 5.8 49 5.8 11 3.8 24 3.8 37 3.8 50 3.8 12 1.9 25 1.9 38 1.9 51 1.9 130.0 26 0.0 39 0.0 52 0.0

Considering the electric field provides the driving force in theelectrophoresis, force that is much higher than the gravity, andconsidering that molecules do not sediment under the gravity due totheir small mass; thus the molecules will always migrate toward thedirection of the resultant between the field force lines of bothelectric fields. Therefore, the TAFE electrode array can be arranged ininverted configuration. That is, the cathodes can be placed at thebottom of the chamber and the anodes at the top, thus, loading thesample plugs in the bottom of the minigel, and provoking the moleculesto migrate along the direction opposite to the gravity. This electrodearrangement will be named ‘inverted TAFE configuration’. Theconfiguration facilitates the placement of the gels in the chamber andavoids the ‘double positioning’ errors of the electrodes and the minigel

As results given by this invention, TAFE electrophoresis chambers areprovided in miniTAFE version, chambers that are wide and have multipleUEZ regions. Each UEZ can be activated at will and are all energizedwith a single power supply. NEZ regions are eliminated from thechambers, because they do not play an essential role in the separationof the DNA molecules. Thus, in said chambers, multiple minigels can beplaced, minigels that can simultaneously separate the DNA moleculescontained in few or many sample plugs, for example, in 10, 20, 30, 40 ormore.

To perform the separation of these molecules, said chambers use theamount of reagents needed to analyze the ‘N’ sample plugs, which containthe molecules that will be separated in ‘t’ time.

The separation between electrodes with opposite polarity is thatdescribed for TAFE minichambers, and for this reason, the chambersseparate the molecules fast.

The chambers are as wide as the equations II–XIII predicted, andaccording to the maximum outputs of the power supplies for pulsed fieldelectrophoresis (see the example in Table 3). For this reason, thechambers are able to separate the molecules contained in at least 52samples of 2.5 mm width.

The chambers have several useful electrophoresis zones (UEZ) that can beused in the experiments or can be occluded and inactivated, the chamberrequires a single power supply and a single system for alternating theelectric fields. For this reason they use efficiently the equipments.

The subdivision of the chamber in several UEZ simulates chambers withvariable width, and makes ‘Nt’ and ‘Bnt’ to vary according to the numberof UEZ used in the chamber (see in Table 2 the example of a chamber with4 ZUE). The volume of buffer solution is replaced as the equations II,III, IV and V predict. For this reason, they can analyze few or manysamples using efficiently the reagents.

The chambers can be constructed in conventional TAFE configuration or ininverted TAFE configuration. They can be made with acrylic, teflon orany other material with high dielectric constant.

The non-useful electrophoresis zones (NEZ) are occluded with pieces ofthe proper shapes, which are made of materials with high dielectricconstant. Otherwise, they could be eliminated from the chambers by meansof any construction procedure.

Several chambers that have the mentioned characteristics can beconstructed We named them type I and type II chambers.

The type I chambers. Type I chambers are the simplest one and, as allthese chambers, they have a small separation between the electrodes withopposite polarity, they are not deep, nor tall, but they are wide. Theelectrodes are as long as the width of the chamber The chambers have anelectrode miniplatform, which can be fixed to the chamber or can beremovable. Type I TAFE chambers can have the cathodes at the top(conventional TAFE configuration) or at the bottom (inverted TAFEconfiguration). In the later case, sample plugs are loaded in the wells,which are in the minigel bottom, thus the molecules migrate along thedirection opposite to the gravity.

The NEZ can be eliminated from the chamber by constructing the frontalwalls with the proper disposition (frontal walls are parallel to theelectrodes), in particular, if the electrodes are arranged in invertedTAFE configuration. To do it, said walls should form a small angle withthe plane containing the anode and cathode located at the same side ofthe minigel. Thus, as said plane does, the walls form an angle with thefloor of the chamber. NEZ are eliminated from chambers havingconventional TAFE configuration by placing in it the pieces of theproper shapes and made of materials with high dielectric constant.

These chambers have several UEZ and support several minigels, which areplaced widthwise, in tandem. To achieve it, frames as width as thechamber can be designed. Said frame is subdivided in less widened framesand all minigels are simultaneously cast in them. Further, the largeframe is slid into the chamber and acts as support of all minigels thatwill be used, thus, allowing their handling. Minigels can besimultaneously cast in those frames, further removed from them, andfinally placed directly into the chamber. To do it, at the center of thechamber must be pieces that are laterally grooved, through which saidminigels slide. The separation between those pieces will be equal to theminigel width, that is, the width of one UEZ. For casting minigels, theframe must be placed between two flat acrylic sheets, which are preparedto place the comb Further, these pieces are clamped. The frame can havelateral notches to fix the comb in a single position.

In turn, each minigel supports a maximum number of samples that dependson its width In this way, chambers having several UEZ that support oneminigel each will be available. Said chambers will be able to separatefew or many samples, using a single power supply and common electrodes.The volume of buffer solution will depend on the number of UEZ used.Thus, all samples that are analyzed in these UEZ are separated in acommon buffer solution, at the same temperature and voltage applied.

According to the above principles, are achieved variability of thenumber of minigels that can be placed in the chambers, of the buffervolume (‘Bnt’) used in each experiment and of the maximum number ofsamples (‘Nt’) that can be simultaneously analyzed in aco-electrophoresis.

The type II chambers. A variant of the chambers, which is proposed inthis invention, avoids the use of very long electrodes. As the type Ichamber, the type II has small distance between opposite electrodes,thus these chambers have little depth and height. However, each UEZ hasits own miniplatform of electrodes, and these miniplatforms are arrangedin tandem and are removable from the chambers. Each miniplatform uses aminigel into which are loaded as many samples as the gel width permits,width, that in turn, depends on the electrode length.

The electrodes of one or several miniplatforms can be energized using asingle power supply. To do it, the electrode arrays of the miniplatformsare connected in parallel, that is, the anodes are pluggedconsecutively, as well as the cathodes. Differing from the type Ichambers, the UEZ (or miniplatforms of electrodes) that will not beactivated in an experiment can be fully occluded using pieces of shapesimilar to the miniplatform Those pieces are made of materials with highdielectric constant. The connection in parallel among the miniplatformsof a chamber warrants the continuity among the electrodes of theseminiplatforms, and permits to perform co-electrophoresis of all samplesloaded in the minigels using a single power supply and commonelectrodes. Thus, all sample plugs are separated in a common buffersolution, at the same experimental temperature. and applied voltage.According to these principles, among experiments, vary the number of UEZactivated in the chamber, the number of minigels used and the volume ofreagents (‘Bnt’). The maximum number of samples (‘Nt’) that can besimultaneously analyzed in a co-electrophoresis also varies among theexperiments.

In type II chambers, the multiple miniplatforms of electrodes could havetheir cathodes at the top (conventional TAFE configuration) or at thebottom (inverted TAFE configuration) In type II chambers havingelectrodes arranged in inverted TAFE configuration, the buffer solutionregions that are crossed by field force lines that do not act on theminigel can be eliminated with the chamber own walls. To achieve this,the frontal walls of the chamber, or walls that are parallel to theelectrodes, should form a small angle with the plane containing thecathode and anode placed at the same side of the gel (or form a smallangle with said plane). Thus, as it happened with said plane, thesewalls from an angle with the floor of the chamber.

In the construction of type II chambers, the miniplatforms can beconnected or activates by any procedure. For example, to place in thelid the wires that connect neighboring miniplatforms, or to place in thelid the plugs alone, thus permitting the wires to be external to thechamber, or to place the connections in the chamber walls or directlyamong the miniplatforms. On the other hand, the miniplatforms can be ofany shape that fits into the chamber, provided they contain theelectrode array in TAFE configuration; whereas the electrodes can bearranged permanently in the chamber or they can be removable. Theregions of the chamber, in which miniplatforms are not going to beactivated, can be eliminated from the experiment using any procedure;placing solid or empty blocks., blocks that are fixed to the walls orfilled with any liquid As it is done in the use of type I chambers, theminigels can be cast in frames.

B. The Obtainment of Reproducible Band Patterns in the Chambers ProvidedIn This Invention.

The chambers provided in this invention are characterized by:

-   -   They have a system to attenuate turbulences of the buffer        flowing throughout the chamber, system that additionally        maintains constant the temperature and the homogeneity of the        composition of buffer during the electrophoresis.    -   They have a system to insert into the electrophoresis chamber        and remove from it the electrodes, system that maintains the        electrodes stretched. This system uses rubber elastic plugs that        are bored and inserted into holes that were drilled in the floor        or the lateral walls of the chambers. The electrodes pass        through the plug bore.    -   They have a system permitting the experimenter to pull tight the        electrodes of the MiniTAFE and multiminiTAFE chambers.

In this invention are also provided a group of accessories that arerelevant to obtain reproducible band patterns. They are:

-   -   An accessory set that permits to cast minigels that have flat        surfaces. The system warrants the required minigel dimensions.    -   An accessory set that permits to align the sample plugs in the        migration origin.    -   An accessory set that permits to prepare sample plugs of        homogeneous sizes        B.1.—Systems for Attenuating Turbulences of the Buffer Flow and        Homogenizing Buffer Conductivity and Temperature in the Chambers

In the neighborhood of the electrodes, the buffer solution changes itsconduction properties due to the electrolysis. This effect isparticularly relevant in CHEF chambers that have a hexagonal array ofmultiple electrodes placed around the minigel For this reason, thevalues of buffer conductivity ‘σ’ in those regions of the chamber candiffer from values in other regions of the chamber. This situation iscritical in CHEF minichambers. The circulation of the electrophoresisbuffer throughout the chamber at high flow velocity is equivalent tostir the buffer solution. Thus, said high flow velocity represents theway to warrant the homogeneity of buffer conductivity throughout theelectrophoresis chamber For instance, the exchanging of the chambervolume in 3 minutes is enough for this purpose.

When the electrolyte circulates at high flow velocity, turbulencesappear in the fluid of the chamber Another possible origin ofturbulences of the flow is that several peristaltic pumps injectcontinuously small volume of buffer, as fluid pulses.

The system developed in this invention to circulate the buffer at highflow velocity, is based on the following principle. The cross sectionarea of the buffer through which the electric current flows must beconstant in the whole chamber.

The constancy of the sectional area avoids the electric current flowingthroughout the chamber to be randomly modified by local changes in thebuffer electrical resistance due to the presence of waves, vortices orturbulences during fluid circulation. The principle is based on the factthat the resistance (R) of any electrolyte filling the electrophoresischamber is determined by:

-   -   The electrolyte conductivity (σ),    -   The separation between the electrodes with opposite polarity        (d),    -   The cross section area that is transversal to the flow of the        electric current (A)

These variables are related according to the formula XV.R=(1/σ)·(d/A)  (XV)

Thus, if ‘A’ varies in different zones of the chamber, ‘R’ differs tooand also the electric current ‘I’.

The system for avoiding turbulences of the buffer flowing through CHEFchambers is composed by:

-   -   two types of rectangular sheets, one of A type and the other of        B type, being both made of any material with high dielectric        constant,    -   sheets, which are as wide as the internal width of the chamber,        being those of A type of 2 cm height at least, and those of B        type of 0.5 cm height,    -   A type sheets, which are separated from the floor of the chamber        a distance of 0 02 to 0.05 cm, and always protrude from the        buffer solution filling the chamber, thus determining that        during buffer circulation throughout the chamber, the buffer can        only flow through the gap formed between the A type sheets and        the floor of the chamber.    -   B type sheets, which are glued to the floor of the chamber and        fully submerged in the buffer solution, thus determining that        during buffer circulation throughout the chamber, the buffer can        only run flowing over the B type sheets. Both types of sheets        being assembled in the chamber regions near the inlet and outlet        tubing system. They are assembled from the inlet or outlet        tubing system toward the electrode array in the electrophoresis        chamber with the following order. A type sheet followed by B        type sheet, and repeating ‘n’ times the sheet pair, being ‘n’ an        integer between 1 and 4, and assembling the last sheet apart        from the electrodes approximately 1 cm, last sheet that has to        be of A type.

In this way, the fluid, pumped from the heat exchanger, crashes againstthe A type sheet when it enters into the chamber, and then it flowsunder this sheet. Afterwards, the fluid crashes against the B type sheetand flows over this sheet. These events are repeated at each pair ofsheets of the system for attenuating turbulence of buffer flow, untilsaid buffer flows into the compartment where the electrodes and theminigel are placed and thus runs across it. Further, the buffer solutionsuffers the same crashing process at the outlet region of the chamberfrom which the buffer is withdrawn to the heat exchanger. In this way,the turbulences that could exist in the fluid are attenuated.

The system for attenuating turbulence of the buffer flowing throughoutthe buffer TAFE chamber is formed by:

-   -   two identical sheets which are of the size of the chamber walls        that are parallel to the electrodes.    -   said sheets that are made of a material with high dielectric        constant, having them a horizontal slot in its third inferior        part.    -   slot which is as long as the electrodes and has 0.3 cm in        height.

The sheets of this system are placed near to the inlet of the buffersolution and near to the outlet of said solution. In that way, thesheets divide the chamber in three compartments. the central one thatcontains the electrodes and the minigel, and the two others, into whichthe tubing for circulation enters or leaves the chamber. Duringcirculation, the buffer solution enters directly into one of thesecompartments, and then flows through the slot into the electrophoresiscompartment. From the electrophoresis compartment, the buffer flowsthrough the slot of the other sheet into the outlet compartment. Fromthis last compartment the buffer returns to the heat exchanger. In thisway, the turbulences that could exist in the fluid surface areattenuated.

B.2.—Set of Accessories to Warrant Homogeneity of the Electric CurrentFlowing Through the Minigel.

Following the above reasoning, it is realized that if the gel, orsupporting medium, where the electrophoresis is done, hasirregularities, it will present to the electric current flow a crosssection area (A) that is different among its distinct regions.Therefore, ‘Rm’ (resistance to the electrical current flow through thegel) has to be maintained constant in all regions of the gel.

The accessory set to cast minigels that have flat surfaces is adisassemblable device composed by:

-   -   A flat base plate.    -   Two frames, one of them with a cavity that is rectangular in        shape and the other with a cavity that is square in shape of        0.35 to 0.5 cm in thickness, having they two notches to fit into        them a comb with long teeth while casting the minigels with its        wells. Being the frame thickness and the inner dimensions of the        cavities, the ones which determine the minigel dimensions that        is going to be cast and used as supporting medium in the        electrophoresis in CHEF or TAFE chambers.    -   two covers, the cover 1 or cover that fits in the frontal part        of the comb, and the cover 2 or cover that fits in the rear of        the comb.    -   A second comb that is similar to the above mentioned, but has        shorter teeth and permits to push the loaded samples into the        minigel wells.

The combs with long teeth, that form the wells in the minigels, arefully plain and continuous with the teeth in their frontal surfaces,whereas in the back surfaces and over the teeth they gain thickness,forming a step. The combs provided herein, have similar teeth withthickness comprised between 0.03 and 0.1 cm, teeth width between 0.15 cmand the minigel width minus 0.3 cm, and lengths of the teeth equal tothe minigel thickness minus 0.15 cm. Thus, when the comb, the frame andthe flat base plate are assembled, the teeth are 0.1 cm apart from thebase plate and the rear step is 0.1 cm higher than the frame. The combswith short teeth are similar to the combs with long teeth, exceptingthat their teeth are 0.2 cm shorter.

The cover 2, or the cover that fits in the rear of the comb, has twoflat sides In one of its end, it has a protruding edge that will fit inthe frame during the assembly of the set The cover 1, or the cover thatfits in the frontal part of the comb, has two flat sides, but one ofthem has a bevel edge in wedge formation.

The set is used in the following way:

-   -   on the flat base plate is placed one of the two frames,        specifically the one that has the size of the minigel that will        be prepared,    -   the legs of one of the combs with long teeth are fitted into the        notches that has the frame in its outer sides, giving as result        that the teeth were separated 0.1 cm from the surface of the        flat base plate,    -   the cover 1, that fits in the frontal part of the comb, is        placed on the frame in front of the comb, with the flat surface        turned to face the frame and with the bevel edge against the        comb,    -   said accessory set is clamped, aided by any procedure, the        covers are pressed against the frame, until the interstices        formed between them are sealed, and then, pouring at the proper        temperature the molten gel, temperature that is between 65 and        70° C. when molten agarose is used,    -   the cover 2, or cover that fits into the rear of the comb, is        placed on the frame, in the back of the comb, introducing the        protruding edge of the cover into the rear step of the comb with        long teeth, and then, the agarose is set to rest until        solidification.    -   the comb with long teeth is removed, and on the wedge-shaped        edge of the cover 1, or cover that fits in the frontal part of        the comb, are placed the plugs containing the immobilized DNA        molecules, plugs that are pushed with the aid of any applicator        to let them slide into the wells.    -   once the sample plugs were loaded into the minigel wells, said        plugs are pushed down the bottom of the wells aided by the comb        with short teeth, being it accomplished by fitting the legs of        the comb into the notches of the frame

Thus, it is warranted to cast minigels that have flat surfaces and nomeniscus, minigel in which all sample plugs were loaded at the sameheight and are evenly separated from the frontal or the rear edge of theminigel. Such results are obtained without the disruption of said plugs.

The accessory set to cast minigels with flat surfaces as well as thesystem to attenuate turbulences of buffer flow avoid that the crosssection area of the minigel varies out of control because of theformation of meniscuses in the sides of the gel or among the wells ofthe minigel.

B.3.—Set of Accessories to Form Sample Plugs of Homogeneous Sizes.

Even the values of ‘R’ are warranted to be constant in the bufferfilling the chamber as well as in the minigel; if the agarose plugs,containing the immobilized DNA molecules, do not have similar dimensionsand are not intact, nor aligned, nor evenly separated from the rear orthe frontal minigel edges, the resulting band patterns will bedistorted.

The accessories to prepare DNA samples immobilized in agarose plugs ofhomogeneous dimensions and sizes similar to the gel wells, into whichthey will be loaded, are composed by:

-   -   sample plug makers, consisting each of a flat impermeable block        thicker than 0 5 cm, block that is made of any material and has        multiple parallel grooves, being each groove of 0.2 cm width and        matching its depth with the thickness of the teeth of a given        comb, depth that can be between 0.03 and 0.1 cm, existing blocks        for all possible thickness of the teeth of all combs that can be        used to form the wells in the gel,    -   a flat, rigid and impermeable sheet of at least 0.1 cm        thickness, which plays the role of the cover of the block,    -   sample plug cutters, each being a bar that is as long as or        longer than the grooves of the block of the sample plugs maker,        said cutters having legs in the ends which confer them an        inverted-U shape, said cutters having several protuberances with        cutting edges In its inferior part, said protuberances        protruding 0.1 cm from the bar, said cutting edges being        transversal to the longest dimension of the bar and 0.2 cm in        length, said cutting edges being evenly spaced a distance that        is from about 0.15 to the gel width minus 0 3 cm,    -   they has a method of using.

The use of these accessories has the following steps:

-   -   an agarose cell suspension is prepared and maintained at 45° C.,        whereas the sample plug maker and its cover are warmed at 45°        C.,    -   said cell suspension is poured into the grooves of the block of        the sample plug maker.    -   the block is covered with its cover and is maintained at room        temperature or lower temperature, until the gel is solidified    -   once the gel is solidified, the sample plug cutter is placed        aligned lengthwise on the first groove, with the protruding        cutting edges turned to face the groove and placed transversally        to the largest dimension of the groove,    -   the sample plug cutter is pressed down,    -   the sample plug cutter is removed, the block is tilted and the        plugs are pushed into a vessel that contains the proper solution        to treat them,    -   the process is repeated for all strips of agarose solidified in        all grooves of the block.

By this procedure, it is warranted that the formed sample plugs weresimilar and had dimensions that coincided with those of the gel wellsinto which they will be loaded into for latter subjecting DNA moleculesto the electrophoresis process.

B.4.—System for Fixing and Pulling Tight the Electrodes to AvoidDistortion of the Equipotential Lines in the Electrophoresis Chambers.

In the invention, it was considered that to avoid random variations ofthe gradient of the electric potential applied to the molecules duringthe electrophoresis, it is necessary that the equipotential lines in thegel were not distorted. That is achieved if the electrodes remainstretched. To attain it, in this invention, the electrodes were insertedinto the chamber through the holes perforated in the floor of the CHEFchambers or in the walls of the TAFE chambers. In these holes are theninserted silicon elastics plugs, and through the plug bores are insertedthe electrodes. Thus, it was warranted that even the electrodes becomethinner because of the use in pulsed field gel electrophoresis, theywill be pressed always by the plug and therefore fixed.

Additionally, in TAFE system the electrodes are long. So, theyoccasionally slacken. To avoid this problem, in this invention, the TAFEchambers were equipped with a system to pull tight the electrodes. Thesystem has.

-   -   a rod with the top side crossed by a slot, slotted rod that is        able to turn and has a waist-shaped notch which was crossed by a        hole,    -   said hole, through which an electrode end is inserted and        further bended around the waist-shaped notch of the rod,    -   a grub screw that sets the slotted rod at the desired position.

This system is placed in the chamber at the exit of the electrode. Theelectrodes are pulled tight by the experimenter according to thefollowing procedure:

-   -   the grub screw, that immobilizes the slotted rod where the        electrode is inserted, is loosened,    -   the slotted rod is turned the required angle to pull tight said        electrode,    -   the grub screw is tightened to set the slotted rod in the        current position and to maintain the electrode stretched.

In this way, non-distorted equipotential lines are warranted widthwisethe vertical gel

In this invention, reproducible patterns are warranted, because anadequate system to energize the electrodes with the proper voltagevalues is used, and the electrodes are maintained stretched;furthermore, systems were used to warrant that the migrations ofany-sized molecules were not perturbed by local transient changes of theelectrical resistance of the buffer or the gel. Such changes provokedistortions of the migration lanes and the bands formed by the moleculesafter the electrophoresis process.

C. Methods for Using the Chambers Provided in this Invention and Methodfor the Calculation and Selection of the Electrophoresis Run Time in theChambers.

In this part of the invention, a method of calculation was created. Itpermits to estimate the electrophoresis running time for differentelectric field, temperature and duration of the electric pulses. Themethod is based on the existence of a set of equations that describesthe migration per pulse ‘m’ of any lineal DNA molecule in CHEFequipments (López-Cánovas L y cols, J. of Chromatogr. A 1998, 806,123–139). These equations are fully incorporated here as reference.m=vr·tp·Γ(tp−tr)+vm·(tp−tr)·[1−Γ(tp−tr)]wherevr=0.0207·[Q·E ¹ ⁴⁵/(8π·η·L ^(1.35))];vm=0 665·[Q·E ¹ ⁷⁶/(8·π·η·L ¹ ⁰⁸)];tr=0.134·(L ¹ ¹⁴ /vr)^(0.926),Γ(tp−tr)=1 if (tp−tr)≦0 and Γ(tp−tr)=0 if (tp−tr)>0.

In these relationships, the variables and parameters have the followingdefinitions

‘tr’ is the reorientation times (s) of a lineal DNA molecule,

‘vr’ and ‘vm’ are the migration velocities (in cm/s) of said moleculeduring and after the reorientation, respectively,

‘Q’ is the net charge of the molecule (in statcoulomb) given by 1e·bp,where ‘e’ is the electron charge and ‘bp’ the base pairs.

‘L’ is the contour length (in cm) of the lineal DNA molecule, given by0.34 nm·bp.

‘E’ is the electric field strength in statvolt/cm,

‘η’ is the buffer viscosity in Poises, calculated by interpolating thevalue of the experimental temperature in a polynomial that relates waterviscosity to the experimental temperature (in ° C.),

‘tp’ is the pulse time duration (s).

To feed the method, the migration per pulse ‘m’ of the smallest moleculeis first calculated.

This is performed:

-   -   by feeding the above relations with the values of the electric        field, temperature and pulse time that will be used in the        electrophoresis,    -   by feeding the above relations with the size, in base pairs ‘bp’        of the smallest DNA molecule to be separated,    -   by calculating ‘m’, provided the electric field and temperature        were comprised between 5.8 and 16 V/cm and between 10–30° C.,        respectively, and assuming that in the electrophoresis process        were used 1.5% agarose gel and 0.5×TBE buffer (1×TBE 89 mM Tris,        89 mM, boric acid 2 mM EDTA, pH 8.3).

Once the migration per pulse is calculated, this value is used to feedthe method to calculate the electrophoresis running time. In saidmethod, the electrophoresis running time (‘te’ in seconds) is calculatedas:te=[(D/m)·2·tp]

The method also requires the distance ‘D’ in centimeters that thesmallest molecule is wanted to migrate in the gel. The preferred valueof ‘D’ is the distance that separates the migration origin and theinferior edge of the gel, minus 0.1 or 0.2 cm. According to the method,for 30° C., the electrophoresis running times, needed to separate DNAmolecules up to 2 Mb, are between 1.5 and 9 hours at 16 and 5.8 V/cm,respectively, whereas for 10° C., they are comprised between 2.5 and14.5 hours at 16 and 5.8 V/cm, respectively

The above mentioned steps warrant the proper use of the chamber and theobtainment of similar band patterns when equal intensities of theelectric field, temperature, electrophoresis running time, bufferconcentration, and electric pulses are applied The method to perform theelectrophoresis process in the chambers of this invention, aided bymentioned accessories and methods, are summarized in the followingsteps:

-   -   the chamber is connected to the electric field switching device,        the chamber is filled with buffer solution and connected to the        external heat exchanger, the proper assembly of the system to        attenuate turbulences of the buffer flow is checked, and the        buffer solution is circulated throughout the chamber until the        desired temperature is reached,    -   with the aid of the accessories to prepare gels having flat        surfaces and using the proper comb, the gels are prepared for        the separation of large DNA molecules, gels that are up to 0.5        cm in thickness in accordance to the selected chamber,    -   into the gel wells are loaded the plugs containing the DNA        molecules that will be separated, molecules that were        immobilized previously in said plugs, being the plug dimensions        similar to those of the gel wells,    -   buffer circulation is interrupted, and the gel loaded with the        plugs, is submerged in the buffer solution, which is at the        desired temperature; the buffer circulation is restored    -   the electrophoresis running time that will separate the DNA        molecules is calculated using the calculation method that        depends on the experimental conditions that will be used, as        well as from the length of the gel in which the electrophoresis        will be done,    -   the system is energized, and the electrophoresis of the DNA        molecules is performed in the gel of flat surfaces, carrying out        the circulation of buffer solution at high flow velocity.

In summary, the chambers provided in this invention are small and haveseparations between their electrodes with opposite polarity thatdetermine the dimensions of the chambers. Although, theseelectrophoresis chambers are small, they used gels that are long enoughto reveal the separation of large DNA molecules in band patternsTherefore, the chambers permit high throughput sample format, fact thatconverts them in a new tool to perform studies that require fastresults, and the comparison of the results given by numerous samples.This process can be done is short time, saving reagents and biologicalmaterial.

In the following sections are shown several examples of the chambers andthe accessories provided in this invention.

EXAMPLES Example 1 Chambers that have Multiple Useful ElectrophoresisZones (UEZ): Type I TAFE Multiminichamber.

In the FIG. 1 is shown an exploded isometric view of a scheme of thechamber 1 In the view is shown the four electrodes 2 of the conventionalTAFE arrangement. The width 3 of the chamber is 316 mm, the height 5 is74 mm and the depth 6 is 114 mm. The frontal 8 and the side 9 walls ofthe chamber are also pointed out. The bottom 18 of the chamber has anexcavation 7 that accommodates the frame 16 carrying the cast minigels20 that the chamber uses. In the side walls 9 are the milled grooves 4through which the frame 16 slides into the chamber. The frame dimensionsare 48 mm tall by 320 mm wide by 5 mm thick. This frame supports fourminigels 20 of 38 mm height and 71.25 mm width The wells 21 for loadingthe samples in the minigels 20 are formed by inserting a comb that hasteeth of 3 mm width and spaced 2 mm.

FIG. 1 shows a three-dimensional scheme of the lid 22, of the blocks 17that are used to occlude the non-useful electrophoresis zone (NEZ) ofthe chamber, and the blocks 15 that occlude the UEZ regions of thechamber.

The FIG. 2 shows the details of the side view of the chamber 1. In theside wall are marked with crosses (+) the end of the electrodes 2, beingthe cathodes placed in the top and the anode in the bottom. Theelectrodes are 316 mm length and are parallel to the frontal wall (8 inFIG. 1) of the chamber. The groove 4, for sliding the frame 16 thatcontains the minigels or the unframed minigels, is excavated in themiddle of the side wall (9 in FIG. 1) and equidistant from the anodes orthe cathodes. The blocks 17 to occlude the NEZ of the chamber, the lid22 and the floor 18 of the chamber 22 are shown as hatched regions. Theouter sides of the blocks 17 are parallels to the frontal walls 8 of thechamber, whereas the inner sides can form a small angle with the planethat contains the anode and the cathode of the same side of the gelThere are blocks 17 as many as UEZ regions in the chamber Blocks 15 areused to occlude the UEZ regions.

Chamber 1 (FIG. 1) has four UEZ regions In the active UEZ regions areplaced the blocks 17 (FIG. 2) to occlude the NEZ regions. To occlude theinactive UEZ regions: the blocks 17 (FIG. 2) are replaced by the blocks15 (FIG. 2) that have rectangular shape. Minigels are not placed in theinactive UEZ regions.

To cast the minigels 20 (FIG. 1), the frame 16 (FIG. 1) is placed on asheet made of acrylic, teflon or other suitable material, and the comb,or several single combs are inserted. Further, the agarose is poured asusually and it is covered with suitable covers To perform theelectrophoresis, the sample plugs are loaded in the wells 21 of theminigels (20, FIG. 1), these minigels are introduced into the chamber(1, FIG. 1), by sliding the frame (16, FIG. 1) through the grooves (4,FIG. 1). The UEZ that will not be used are occluded with the blocks (15,FIG. 2) and in the UEZ that will be used the blocks (17, FIGS. 1 y 2)are placed. The chamber (1, FIG. 1) is filled with buffer solution andthe electrodes (2, FIG. 1) are energized through the electric fieldswitching unit by means of using a power supply. To maintain constantthe temperature, cold buffer solution is circulated. The inlet andoutlet tubing used for the cooling of the buffer solution are insertedin the frontal walls (8 in FIG. 1) of the chamber 1.

FIG. 3 shows the 52 band patterns 24 given by S. cerevisiae chromosomesin the four minigels of the chamber (1, FIG. 1). These patterns wereobtained at 8.33 V/cm, 15° C., in 1.5% agarose gel, 0.5×TBE buffersolution, 12 hours of electrophoresis time and at 80 seconds of pulsetime duration. The minigels were cast in the frame (16 in the FIG. 1) asit was above described.

Based on experiments done in the chamber (1, FIG. 1) in 0.5×TBE buffersolution, 15% agarose (Lachema) gel, using one, two, three or the fourUEZ regions, and maintaining constant the height of the buffer in thechamber; for the equation IV was obtainedC(vessel)=a ₀ +a ₁(d/L)^(0.1)where a₀=−0.786 and a₁=1.047 and have variances of 1.451·10⁻⁴ y 16949·10⁻⁴, respectively. Both coefficients differed significantly fromzero. For the equation VIII, it was additionally obtainedRp ⁰ ⁵=−1.522+Re·2.1096·10⁻²+87.³¹ /V _(DC)+Temperature·2.2697·10⁻²

The coefficients of the equations were calculated by measuring ‘V_(DC)’and ‘I_(DC)’ in the chambers. It permitted to estimate ‘I₀’ and themaximum values of E that can be applied in MultiMiniTAFE chambers (Table3). These results are calculated to the power supplies most used inPFGE. This procedure was used to select the dimensions of the chambersthat were constructed. As it was expected, the electrolyte polarization‘I₀’ does riot linearly depend on the electric field.

For the buffer exhaustion constant, it was obtained:k=−3.6365·10⁻²+Field·1.6135·10⁻²

On the other side, according to the equations fitted, if a power supplywith a maximum output of 200 Watt and 0.4 A is used, and are also usedthe four UEZ of the chamber and 20° C., then, the values of ‘E’ near to10 V/cm demand the replacement of the buffer solution every one hour.Thus, indicating that the use of the four UEZ regions is not efficientat said electric field values. In the example of the FIG. 3, the bandpatterns of the S. cerevisiae chromosomal DNA of the 52 samples plugswere obtained in only 12 hours, but 1 L of buffer solution had to bereplaced after the first 7 hours of electrophoresis. That time coincideswith the time predicted by the equations.

TABLE 3 Maximum electric field intensities (‘E’) that can be set in TAFEmultiminichambers of different widths, using several powers supplies forPFGE. Power Width E volt/cm E volt/cm E volt/cm E volt/cm E volt/cmsupply Cm T = 10° C. T = 15° C. T = 20° C. T = 25° C. T = 30° C. Imax =0.4 10 25.8 22.6 19.9 17.4 15.4 Vmax = 500 20 17.4 15.4 13.5 11.9 10.6Pmax = 200 30 13.0 11.5 10.3 9.1 8.1 40 10.3 9.1 8.1 7.2 6.4 50 8.2 7.36.5 5.8 5.3 Imax = 1.0 10 49.6 46.4 43.5 40.8 37.9 Vmax = 600 20 40.637.9 33.5 29.5 26.0 Pmax = 300 30 32.3 28.6 25.3 22.3 19.9 40 25.3 22.419.9 17.7 15.7 50 20.3 17.9 15.9 14.2 12.6 Imax = 2.0 10 38.3 38.3 38.338.3 38.2 Vmax = 300 20 38.3 38.2 35.8 33.6 31.7 Pmax = 300 30 35.3 33.131.2 29.2 27.6 40 31.2 29.3 27.6 26.0 24.5 50 27.8 26.2 24.6 23.3 21.9

A variant of said chamber that does not use the blocks (17, FIG. 1) toocclude the NEZ can be designed. Its advantages and drawbacks are as forthe chamber mentioned in the above paragraph, but the variant useslarger amount of reagents and the electric current, and consequently,the power generated in it, are higher. Nevertheless, the time requiredto exhaust the buffer solution is longer. Variants of these chambers canbe also designed in inverted TAFE configuration. The design of thechambers that have electrodes arranged in inverted TAFE configuration isshown in the example of the type II chamber.

‘E’ was estimated using the equations II, III, IV y V. Imax maximumelectric current output (in Ampere) of the power supply, Vmax: maximumvoltage output (in volt) of the power supply, Pmax: maximum power output(in Watt) of the power supply. The values of ‘E’ were estimated for the85% of Imax, Vmax and Pmax of the power supply used.

According to the above principles, it is achieved that the number of UEZthat could be activated in the chamber, the number of minigels used inan experiment, and the volume of reagents (‘Bnt’) used in eachexperiment can vary. The maximum number of sample plugs (‘Nt’) that canbe analyzed per co-electrophoresis is also variable.

Example 2 Chambers that have Multiples Useful Electrophoresis Zones:Type II TAFE Multiminichamber.

FIGS. 4–7 show several views of a type II chamber that has 3 removablemini-platforms of electrodes.

The FIG. 4 shows a view in exploded form of a side section of thechamber 34, the removable electrode mini-platform 25, the frame 30 thatsupports the gel 31 and the sample plugs 36. In the mini-platform 25,the cathodes 26 are at the bottom of the chamber, whereas the anodes 27are at the top (inverted TAFE configuration). The outer walls 28 play arole similar to the blocks 17 of the type I chambers (FIGS. 1 and 2), ie said walls eliminate the NEZ regions. In the middle of the electrodemini-platform is located the groove 29 through which is slid the frame30 that contains the minigel 31 of the mini-platform. The pieces 40 ofthe mini-platforms 25 have the ducts 41 through which the tubing pass tocirculate the buffer solution throughout the chamber.

It is also shown the frontal walls 33 of the chamber 34 where themini-platforms 25 may be optionally placed. In the chamber 34, themini-platforms walls 28 have a slot 32 to communicate the buffersolution circulating throughout the chamber. During the assembling ordisassembling of the mini-platforms, the pieces 40 are slid into thegrooves 35 milled in the frontal and rear walls 33 of the chamber 34.

FIG. 5 shows a top plan view of the chamber 34, which has assembled thethree mini-platforms of electrodes 25.

FIG. 6 illustrates a top plan view of the chamber 34 and some detailsdescribed in the above figures. In the view is schematically shown thata single electrode mini-platform 25 was assembled in the chamber. Theothers two regions, where the two others mini-platforms could be placed,are shown occluded with the pieces 42 constructed with a material ofhigh dielectric constant.

FIG. 7 shows the top plant view of the lid 55, the connecting ends 43and 45 and the electric connections 44 and 46. The cathodes (26 in FIG.4) of the three mini-platforms are connected in parallel through theconnecting ends 43 and the wires 44, while the anodes (27 in the FIG. 4)are connected in parallel through the connecting ends 45 and the wires46. In this way, the electrodes of all mini-platforms are coupled. Inthis chamber, each mini-platform has its frame 30 to hold the gel 31(FIG. 4). The sample plugs (36 in FIG. 4) are placed in the lower partof the gel, because the electrodes are arranged in inverted TAFEconfiguration.

To perform the electrophoresis in this chamber, first it is decided howmany mini-platforms 25 (FIG. 4) will be activated, and the rest areoccluded or inactivated with the pieces 42 The minigels 31 are cast in away similar to the procedure performed in the type I chamber, and theyare loaded with the sample plugs. Further, the frames containing theminigels and sample plugs are placed in the mini-platforms. Said framescan be placed in the chamber before or after the buffer solution isadded. Once the process is completed, the lid is connected and theelectrodes energized through the electric field switching unit connectedto a power supply.

Example 3 Chambers with Single Useful Electrophoresis Zone: MinichamberCHEF.

In the FIG. 8 is shown a scheme of minichamber CHEF. Inside a hexagonalarray 60 of eighteen electrodes is placed a gel 61 of agarose or othermaterial able to polymerize and form a matrix. The gel 61 is retained inits position with the squares 62 glued to a base plate 62 that isintroduced into a depression 69 excavated in the floor of the chamberInto the gel 61, are loaded the sample plugs 64 which are formed of thesame material of said gel and contain immobilized chromosome-sized DNAmolecules. Sample plugs 64 are loaded in such a way that, after settingthe electric field at the selected intensity and switching its directionof application, the molecules are separated according to their sizes,giving as results reproducible band patterns among the different lanesof the gel. The chamber is filled with the buffer solution to permit themolecules to migrate.

The temperature, pH, concentration and others parameters of the solutionshould be homogeneous and constant throughout the chamber and theelectrophoresis process For this reason, the buffer is exchangedcontinuously with an extra buffer volume that is contained in athermostatic recipient.

To achieve buffer homogeneity, it is important to circulate said buffersolution at a high flow velocity. The buffer is added to the chamberthrough the inlet 65 and recovered from it through the outlet 66. Infront of the inlet 65 and the outlet 66 is located a system 67 forattenuating turbulences of the buffer solution flowing throughout thechamber In the figure, are indicated the two sheets of A type 67 thatwere disassembled to make evident the B type sheets placed in the floorof the chamber. Turbulent flow of the solution affects the electricfield homogeneity through the chamber and provokes band patterndistortion Some physical dimensions of CHEF minichambers are presentedin table 4 The information does not limit the scope of this invention,but it illustrates the chambers disclosed here.

TABLE 4 Real parameters of some CHEF minichambers. Chamber MiniCHEF 1MiniCHEF 2 MiniCHEF 3 Separation between electrodes with 11.6 6.2 11.6opposite polarity (cm) Number of electrodes 18 18 36 Area of thechamber's floor (cm²) 272 94 272 including the system for attenuatingthe turbulence buffer flow Volume of the buffer solution in the 225 80225 chamber (ml) Dimensions of the square gel (cm) 4 × 4 × 0.5 2.2 × 2.2× 0.2 4 × 4 × 0.5 Dimensions of the rectangular gel (cm) 7 × 4 × 0.5 3.6× 2.2 × 0.2 7 × 4 × 0 5 Number of sample plugs of 0.15 cm width 27 13 27loaded into the rectangular gel

Example 4 Chambers with Single Useful Electrophoresis Zones: TAFEMinichamber.

In FIG. 9 is shown a scheme of TAFE minichamber with an invertedelectrode configuration The gel 71, which could be of agarose or othermaterial able to polymerize to form a matrix, is placed vertically inthe mid-way between the two positive electrodes 72 or the two negativeelectrodes 73. Sample plugs, 74 containing DNA molecules, are loaded insuch a way that, after setting the electric field at the selectedintensity and switching its direction of application, the molecules areseparated according to their sizes, giving as results straight bandpatterns. The chamber is filled with the buffer solution to permit themolecules to migrate. The buffer is added to the chamber through theinlet 75 and recovered from it through the outlet 76. In front of theinlet 75 and the outlet 76 is located a system 77 for attenuating theturbulences buffer flowing throughout the chamber Some physicaldimensions of TAFE minichambers are presented in table 5 The informationdoes not limit the scope of this invention, but it illustrates thechambers disclosed here.

TABLE 5 Real parameters of some TAFE minichambers. Chamber MiniTAFE 1MiniTAFE 2 Separation between electrodes with opposite 7.8 10 polarity(cm) Area of the wall into which the electrodes are 127.7 166.8 fixed(cm²) Dimensions of the chamber (cm) 15.2 × 7.1 × 8.4 20.1 × 6 × 8.3Volume of the buffer solution (ml) 530 800 Dimensions of the gel (cm) 7× 4 × 0.5 6.3 × 5.2 × 0.4 Number of sample plugs of 0.15 cm width 27 24

Example 5 Electric Parameters of Some Minichambers of this Invention.

In this chamber, the separation between the electrodes with oppositepolarity (equal or less than 15.0 cm) permits to set the electric fieldstrengths up to 25 V/cm in TAFE and 16 V/cm in CHEF, if 0.5×TBE (1×TBE:89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8 3) is used and voltageless than 375.0 V is set using a power supply with a maximum output of300 Watt. The electric resistance in these chambers is several thousandOhm, because the small volume of buffer solution used. Because of this,high electric field strength can be achieved using a power supply withlow maximum output.

The electric parameters and the power consumption of the chambersprovided in this invention are shown in table 6. Measurements were doneusing the volumes of 0.5×TBE described in tables 4 and 5 at 20° C.

TABLE 6 Electric parameters of CHEF and TAFE minichambers. ChamberMiniCHEF 1 MiniTAFE 1 Electric field strength (V/cm) 10 16 8 20 Voltageset (V) 116.0 185.5 62.4 156.0 Electric current intensity (mA) 85.0139.9 63.4 168.0 Generated power (Watt) 9.8 26.0 4.0 26.2

Example 6 Way of Fixing the Electrodes in CHEF and TAFE Chambers

The fixing of the electrodes in their positions in CHEF minichambers andin TAFE minichamber and TAFE multiminichambers are shown in FIG. 10.

The electrodes are a platinum wire 81 that has an approximate diameterof 0 05 cm They communicate the electric energy from an externalelectric circuitry to the solution contained in the chambers, thusimposing the electric field that provokes the migration and separationof DNA molecules.

The floor 82 of CHEF chamber and the two sides walls 83 (the walls thatsupport the gel) of TAFE chamber are bored to permit to insert into themthe platinum wire forming the electrode. To fix the electrodes in theholes and avoid the leaking of the buffer solution, the wires 81 areinserted in the bore of an elastic plug 84. The elastic plug 84 shouldbe made of a very flexible material that fit into the hole and the wire81, even when said wire has become thinner due to the use.

Example 7 Example of a Set to Prepare Gels that Have Flat Surfaces.

A crucial element to obtain straight and reproducible band patternsusing these chambers is the shape of the gels 61 and 71. The surfaces ofsaid gels should be flat, otherwise the homogeneity of the electricfield in the gel will be affected and distorted band patterns will beobtained. A rear view of the accessories used to cast the gels 61 and 71is shown in FIG. 11.

Gels 61 and 71 are cast on a base plate 91 of flat surfaces, which islarge enough to support the frame 92. The frame thickness 92 willdetermine the gel thickness that will be cast. The dimensions of theinternal cavity of flat surfaces 93 will determine the width and thelength of the gels 61 and 71.

In the outer sides of the frame 92, are the notches 94. They are near toan edge of the frame 92 and evenly separated from such edge. The legs 96of the comb 95 will be fitted into the notches 94, then the notches 94are as wide as the legs 96.

The comb 95, has teeth 97 with bevel cuts to make equal theircross-sections to those of the sample plugs 64 and 74. The comb 95 isflat in the frontal part whereas in the rear over the teeth 97 it gainsthickness forming a step, being the length of the teeth equal to theframe thickness 92 minus 1.0 mm. The legs 96 are of this length. In FIG.11 is shown the magnification of one of the teeth 97 to appreciate thestep.

The cover 100 has flat surfaces and one of its edges has a bevel cut inwedge formation 101. A magnified view of part of the bevel edge 101 isshown. The width of the cover 100, at least in the bevel edge 101, islarger that the width of the cavity 93 of the frame 92 The cover 103also has flat surfaces, excepting the edge that has the protruding end104 of 0.1 cm thickness. A magnified view of part of the protruding end104 is shown. The protruding end 104 is wider than the cavity 93 of theframe 92, but it is shorter than the distance between the internal facesof the legs 96. The comb 105 is similar to the one 95, but its teeth 106are 0.2 cm shorter.

To cast the gel, the base plate 91 is placed on a horizontal surface,the frame 92 is placed on said surface, with the notches near the backof the base plate 91. The comb 95 is fitted into the notches 94 with theteeth 97 turned forward. After that, the cover 100 is placed on theframe 92 in front of the comb 95 with the flat surface turned to facethe frame and the bevel edge 101 against the comb. The arrows indicatethe direction of accessories assembly The accessory set is immobilizedby fixing with clamps, or other device, the frame 92 to the base plate91. The agarose or other solution able to polymerize is poured behindthe comb 95. The temperature of the molten agarose is from 65 to 70° C.For other solution the temperature can vary. The volume of molten gel,to be added, should be enough to fill to the brim of the cavity formedamong the base plate 91, the walls of the cavity 93 of the frame 92 andthe cover 100. Further, the cover 103 is placed on the frame turning theflat face downward and the protruding end 104 against the back of thecomb 105. It removes the remaining molten gel. The assembled accessoryset is left to set until the molten gel solidifies.

After the gel 61 or 71 was cast, the comb 95 is removed and the sampleplugs 64 or 74 are loaded in the wells 107 which were formed afterremoving the comb. As the cross-sections of the teeth 97 and the sampleplugs 64 and 74 are equal, the sample plugs are uniformly loadedwidthwise the gel and evenly separated from the edges of said gel 61 or71. When the sample plugs 64 and 74 were already loaded, they arefurther pushed into the gel 61 or 71 with the aid of the comb 105 toplace all plugs at similar depth.

The suitable use of the accessories described in this invention permitsto cast gels of flat surfaces and the alignment of the sample plugscontaining immobilized DNA molecules This is one of the necessaryconditions to obtain straight and reproducible band patterns

Example 8 Gel of the CHEF Minichambers and Placement of them in theMinichambers

Gels of different sizes can be placed in CHEF minichambers. To retainthe gel 61 (FIG. 12) in a given position during the electrophoresis, aplastic or acrylic rectangular base plate 63 is used. Square flanges 62,are placed on the base plate 63. Each square flange 63 is placed at eachcorner of a rectangular or square contour where the gel 61 will beretained. The separations between the square flanges 62 are as the samedimensions (length and width) of the gel 61 that will be placed on saidcontour. The maximum height of the square flange 62 should be 0.2 cm, toavoid the attenuation and distortion of the electric field generated inthe chamber

The base plate 63 is inserted into the floor of the chamber in thecenter of the electrodes 60 array In this region of the chamber, thereis an excavation 69 with a rectangular shape of dimensions as those ofthe base plate 63. The depth of the depression 69 is equal to thethickness of the base plate 63, then the gel 61 will be retained at thesame level of the chamber floor. The base plate 63 has several notchesin the edges and in the corners to facilitate its extraction from theexcavation when the experiment is finished. Excepting for the positionof the square flanges 62, all the base plates 63 are identical. Then,gels 61 of different sizes can be used in the same chamber. It isimportant that the set warrants that the gel 61 is well positionedduring the electrophoresis to obtain straight and reproducible bandpatterns.

To perform the electrophoresis, the gel 61 loaded with the sample plugs64 that contains the DNA molecules (said gel prepared with theaccessories described in the example 7). is taken and placed on the baseplate 63 between the four square flanges 62. After that, the base plate63 holding the gel 61 is inserted into the excavation 69 of the floor ofthe chamber. Because DNA molecules acquire negative charge in solutionat neutral pH, they migrate toward the anodes, then, the base plate 63should be placed with the sample plugs 64 nearer to the cathode. Whenthe electrophoresis ends, the gel 61 is removed from the chamber tostain the DNA molecules and visualize the band patterns To clean thechamber or use a different sized gel 61, the base plate 63 is removed byinserting a bar into the notches 111 and levering the base plate 63.

Example 9 System to Pull Tight the Electrodes of the TAFE Minichambers

The electrodes of TAFE minichambers are slacken as they are used. In theFIG. 13 is shown a device to pull tight the platinum wires 81 that formthe electrodes. The slotted rod 115 has a waist-shaped notch 116 crossedwith a hole 117 that has a diameter slightly larger than the platinumwire 81. The wire end 81 is inserted into the hole 117 and the slottedrod 115 is turned with a screwdriver placed in the slot 118 until thewire 81 is pulled tight. To fix the position of the slotted rod 115 agrub screw 119 is loosened before the wire 81 is pulled tight and thenthe grub screw is tightened again.

Example 10 System for Attenuating Turbulences of the Buffer FlowingThroughout the CHEF Minichambers

CHEF minichambers have a system to attenuate turbulences of the bufferflowing throughout the chamber, thus permitting the circulation of thebuffer at high flow velocities. In the FIG. 14 is shown a scheme of thesystem to attenuate turbulences of the buffer flowing throughout theCHEF chambers. It is formed by impermeable A type sheets 121 and B typesheets 122 that are made of a material with high constant dielectric toavoid perturbations of the applied electric field.

The type A sheets 121 are the tallest, and are placed separated from thefloor of the chamber to avoid the buffer to overflow them and forcingthe solution to flows under them The B type sheets 122 are the shortest,they are glued to the chamber and surpass the gap formed between A typesheets 121 and the floor of the chamber.

The A type 121 and B type 122 sheets are placed alternately, beginningand ending with A type sheets 121 and placing between them B type sheets122. The set of A 121 and B type 122 sheets are placed in front of theinlet 65 and outlet 66. The pairs of A 121 and B type 122 sheets can berepeated as many as times as desired until the last is 1 0 cm apart fromthe electrodes.

The buffer solution is injected through the inlet 65 and flowsalternately under A type sheets 121 and over the B type 122. This biasedtrajectory (pointed out by the arrows) will attenuate the variations ofthe fluid pressure provoked by the injection, when the buffer flows overthe gel 61, it almost runs at constant velocity with the absence ofturbulent flow The same process takes place in the other side of thechamber, from which the solution is recovered through the outlet 66.

Example 11 System for Attenuating Turbulences of the Buffer FlowingThroughout the TAFE Chambers.

The TAFE chambers that have single or multiple UEZ also have a system toattenuate the turbulences of the buffer flowing throughout the chamber,thus permitting the circulation of the buffer at high flow velocity.

In the FIG. 15 is shown a detailed scheme of the system to attenuate theturbulences of the buffer flowing throughout a TAFE minichamber with asingle UEZ.

The system is made of impermeable sheets 131 with high dielectricconstant. They avoid the free buffer flow throughout the chamber,excepting through the slots 132. The buffer solution is injected throughthe inlet 75 and it is discharged through the outlet 76. The fluidpressure changes produced during the injection and discharge of thebuffer from the chamber are attenuated in the cavities 133, so when thebuffer flows through the gel 71, it almost runs at constant velocitywith the absence of turbulent flow. The arrows indicate the trajectoryof the buffer from inlet 75 to the outlet 76

The maximum velocity of flow in these chambers that does not provoketurbulent flow of the buffer depends on the size and the volume of thechambers. The maximum flow velocities that could be used in the chambersdisclosed in examples 3 and 4, and do not perturb the solution are shownin table 7

TABLE 7 Maximum flow of circulation that does not provokes turbulentflow of the buffer solution filling PFGE minichambers. Maximum flowvelocity with Buffer exchange Volume time the absence of turbulent flow(minutes). Chamber (ml) (ml/minute). MiniCHEF 1 225 100 2.25 MiniTAFE 1530 280 1.89 MiniCHEF 2 80 44 1.82

Buffer exchange time refers to the time that has to elapse until onechamber volume of buffer must be exchanged.

Example 12 Accessory Set to Prepare the Sample Plugs

As above stated, to obtain reproducible band patterns it is essential tohave samples plugs with identical shape, dimensions and DNAconcentration. Said sample plugs must have dimensions and shapes similarto those of the wells of the electrophoresis gel.

In the FIG. 16, is shown one of the accessory sets designed to obtainsample plugs with said characteristics. Such set has the sample plugapplicator 141, the sample plug handler 142, the sample plugs maker 143,its cover 144 and the sample plug cutter 145.

In the example, the sample plugs maker 143 is an acrylic, rubber orsilicon rectangular block (7×6.9×1 cm length×width×depth) with smoothand flat surfaces, excepting one of said surfaces that has elevenparallel and rectangular grooves 146 excavated on it Said grooves arespaced widthwise of the block in such a way, that the width of thegroove coincides to the height of the sample plugs 148 and the depth ofthe groove 146 coincides to the thickness of the sample plugs 148.

The cover 144 is a glass or acrylic rigid and flat sheet that is placedon the grooved surface of the sample plug maker 143. Pieces 143 and 144are kept together to warrant the airtightness between the differentgrooves 146. Using a pipette an agarose cell suspension is dispensedinto each end of the said grooves. The grooves are well filled and theset is left to set until the agarose solidify. To remove the cover 144,said cover is slid transversally to the grooves 146, in order to avoidthe dragging of the solidified agarose strips 147. The strips 147 arecut in small sample plugs 148 with the cutter 145. To do it, the cuttingedge of the protuberances 149 are placed on each groove and presseddownward to the bottom side of each groove 146. The distance between theprotuberances of the sample plugs cutter determines the width of thesample plugs 148, so it is warrant that the sample plugs 148, containingthe DNA molecules, have the same shape and dimensions.

Once the samples plugs 148 were cut, the sample plug applicator 141 isused to drag said sample plugs through the grooves 146 and then, to letthem fall into the vessels containing the solutions to treat the cells.The sample plug applicator 141 is also used to load the sample plugs 148into the wells 107 (FIG. 11) of the electrophoresis gel. The sample plughandler 142 is used to recover the sample plugs from the vessel wherethe sample plugs are treated or stored.

Example 13 Saccharomyces cerevisiae Chromosomes Band Patterns Obtainedin TAFE Minichamber (With a Single UEZ)

In the FIG. 17 is shown an example of an electrophoresis done in a TAFEminichamber that has 7.8 cm of separation between the electrodes withopposite polarity This minichamber uses a gel 151 that is 7.0 cm widthand 4.0 cm length. In the gel 151, thirteen sample plugs 152 wereloaded, which were 0.25 cm width, 0.07 cm thick and 0 2 cm depth.Saccharomyces cerevisiae intact chromosomal DNA contained in the sampleplugs 152 were separated during the electrophoresis in the band patterns153 in each lane of the gel 154. Each band pattern has eleven bands. Therunning conditions were 60.0 seconds of pulse time, 7.0 hours ofelectrophoresis, 1.5% agarose, 0.5×TBE, 20° C. and 10.0 V/cm. Gelstaining was performed with ethidium bromide.

The above results indicate that TAFE minichamber give a rapid andadequate separation between the bands of the patterns, as well asreproducible patterns between the lanes of the gel.

Example 14 Reproducibility of the Band Patterns Obtained in CHEFMinichambers

The results of three electrophoresis runs done in a CHEF minichamberthat has 11.6 cm of separation between the electrodes with oppositepolarity are shown in the FIG. 18 That minichamber uses square gels 161,162 y 163 of 4.0 cm width. Into the gels 161, 162 and 163 seven sampleplugs 164, 165 and 166 of 0.25 cm width, 0 07 cm thickness and 0 2 cmdepth were loaded. Saccharomyces cerevisiae intact chromosomal DNA,contained in the sample plugs 164, 165 and 166, were separated duringthe electrophoresis in the band patterns 168, 169 and 170 in the lanes171, 172 and 173 of the three gels 161, 162 and 163. The runningconditions were 50.0 seconds of pulse time, 3.5 hours ofelectrophoresis, 1.5% agarose, 0.5×TBE, 20° C. and 9.82 V/cm. Gelstaining was performed with ethidium bromide.

In the figure, it can be observed that the patterns obtained in the gels161, 162 and 163 have the same number of bands in all lanes. Besides,each band 168, 169 and 170 migrated the same distance in the seven lanes171, 172 and 173 of any of the gels 161, 162 and 163. On the other hand,in each lane 171, 172 and 173 of the three gels 161, 162 and 163, thesame electrophoresis band pattern is observed. Said band patterns havethe same number of bands 168, 169 and 170, indicating that theminichamber gave reproducible results in different experiments in veryshort times (3.5 hours)

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 to 3 are schematic diagrams of type I TAFE chambers andelectrophoresis patterns.

FIG. 1 is an exploded view of the three-dimensional scheme of a type ITAFE chamber that has its electrode array in conventional TAFEconfiguration, the grooves through which the gel frame, supporting allminigels and the samples of the four UEZ of the chamber, is slid.Three-dimensional schemes of the frame, minigels, and the cover carryingthe blocks that eliminate NEZ or UEZ regions are also shown.

FIG. 2 is a side view of a scheme of a type I TAFE chamber. The blocksthat eliminate the NEZ, the electrode array in conventional TAFEconfiguration, and the blocks that occlude the UEZ that are not going tobe used in the electrophoresis are shown

FIG. 3 shows the electrophoresis band patterns given by S. cerevisiaechromosomes when they were separated in the four minigels used in thetype I TAFE chamber of the FIG. 1. The molecules were separated at 8.33V/cm, 15° C., during 12 hour of electrophoresis in 1.5% agarose gel,0.5×TBE buffer solution and at 80 seconds of electric pulse duration.After 7 hours of electrophoresis, one liter of buffer solution wasreplaced.

FIGS. 4 to 7 are schemes of the distinguishing features of the type IITAFE chambers

FIG. 4 is an exploded view of a side section of the type II TAFEchamber. One of its miniplatforms of electrodes arranged in invertedTAFE configuration, its gel frame and its gel, as well as the placementof the samples in the bottom of the gel are shown. The electrodeminiplatforms are removable.

FIG. 5 is a plant view of a scheme of the chamber that has 3miniplatforms of electrodes. The three miniplatforms were placed in thechamber.

FIG. 6 is a plant view of a scheme of the chamber that has threeminiplatforms of electrodes. One miniplatform was placed in the chamberand the others were occluded using pieces of the proper shape andmaterial.

FIG. 7 is a plant view of a scheme of the chamber lid. The electricalconnections are shown

FIG. 8 is an exploded isometric view of a scheme of a CHEF minichamberThe chamber, its electrode array and the lid of said chamber are shown.The base plate and the flanges of the square gel are also shown, as wellas the gel and a hypothetic sample plug. The A type sheets of the systemfor attenuating turbulences of the buffer solution flowing throughoutthe chamber are shown disassembled. In the floor of the chamber isappreciated the B type sheet.

FIG. 9 is an isometric view of a scheme of a TAFE minichamber that hasthe electrodes arranged in inverted TAFE configuration. In the figure,the frontal wall of the chamber was drawn transparent to reveal thedetails inside the chamber. In the center of the chamber is observed thegel surrounded by the four electrodes. Beside the electrodes are shownthe slotted sheets of the system for attenuating turbulences of thebuffer solution flowing throughout the chamber.

FIG. 10 shows the way to fix the electrodes in the walls of TAFEminichambers or in the floor of CHEF minichambers. In the upper part, across section view of a region of the CHEF floor is shown, whereas aregion of the TAFE wall is shown in the lower part of the figure. Theelectrodes inserted into the bore of the silicone plugs are also shown

FIG. 11 is a rear view of a scheme of the accessory set to cast gelswith flat surfaces as well as the comb to align the plugs in the gel. Inthe lower part of the figure is observed the base plate to cast the gel,above it is a gel scheme with its wells and a hypothetical sample plug.Above the gel are the frontal cover of the frame, the frame and itsnotches, and the rear cover of the frame. In the upper part of thefigure, are shown schemes of a comb to form the gel wells and the combto align the sample plugs in the gel. Arrows indicate the direction ofthe accessories assembling.

FIG. 12 shows in the left upper part, a scheme of the base platecarrying the square gel In the right upper part, the base plate carryingthe rectangular gel is shown. In the lower part, a top view of thescheme of the CHEF minichambers is shown. Here, are also shown thehexagonal electrode array and the excavation where the base platescarrying the gels are placed.

FIG. 13 shows a scheme of the device to pull tight the electrodes ofTAFE minichambers. In the left part (A) is shown the slotted rod havingloosened the grub screw as well as the platinum wire end at the entry ofthe slotted rod hole. The arrows indicate the direction for piecesassembling. In the right part (B) is shown the slotted rod with theelectrode end inserted into it and coiled around its waist, the slottedrod already rotated, and the grub screw tightened.

FIG. 14 is a side view of a scheme of the system for attenuatingturbulences of the buffer solution flowing through CHEF minichambers, inwhich the wall was drawn transparent The horizontal gel is observed inthe middle of the figure. Immediately, at both sides of the gel areplaced alternately the A and B type sheets. The arrows indicate theflowing of buffer solution throughout the electrophoresis chamber.

FIG. 15 is a side view of a scheme of the system for attenuatingturbulences of the buffer solution flowing throughout TAFE minichambers,in which the wall was drawn transparent. The vertical gel is observed inthe middle of the figure. At both sides of the gel are the sheets ofsaid attenuation system. The arrows indicate the flow of the buffersolution throughout the electrophoresis chamber.

FIG. 16 shows a scheme of the accessory set to form homogeneously sizedsample plugs containing immobilized DNA. In the lower part of the figureis presented the sample plug handler, and immediately above it, thesample plug applicator. Above them is the block of the sample plug makerdisplaying its lengthwise grooves, and are several strips of solidifiedagarose that were already cut to form plugs. Intact strips of solidifiedagarose are also shown. The cover of the block is shown above. In theupper part of the figure are presented the sample plug cutter andseveral plugs that were already cut.

FIG. 17 shows the electrophoresis patterns obtained in a TAFEminichamber. The chromosomes of intact DNA samples from Saccharomycescerevisiae were separated, they were previously immobilized in thirteenagarose plugs. Electrophoresis conditions, 60 of pulse time, seven hoursof electrophoresis, 1.5% agarose, 0.5×TBE, 20° C., 10 0 V/cm. The gel is4.0 cm length and 7.0 cm width. Gel staining was done with ethidiumbromide.

FIG. 18 shows the electrophoresis patterns obtained in three differentexperiments in a CHEF minichamber. In each experiment were separated thechromosomes of intact DNA samples of Saccharomyces cerevisiaeimmobilized previously in seven agarose plugs. Electrophoresisconditions: 50 s of pulse time, 3.5 hours of electrophoresis, 1 5%agarose, 0.5×TBE, 20° C., 9.82 V/cm. The square gel of 4.0 cm length wasused. Gel staining was done with ethidium bromide.

ADVANTAGES OF THE PROPOSED SOLUTIONS

Chambers for pulsed field electrophoresis, methods and accessoriesdisclosed in this invention have the following advantages:

-   -   1—Chambers saving laboratory bench space and chemical and        biological reagents are provided. That is, they use a small        amount of buffer solution and biological sample    -   2—A method for constructing CHEF and TAFE minichambers, casting        the gels and calculating the volume of buffer solution to be        used is provided. The method only needs to be fed with the        separation between the electrodes with opposite polarity    -   3—Although the buffer circulates throughout the chambers at high        flow velocity and the chambers are filled with a small buffer        volume, the band patterns are reproducible, because a system is        provided for attenuating turbulences of the buffer solution        flowing throughout the chambers.    -   4—The chambers are small, thus, high electric fields can be        applied using power supplies of low power output, so the        electrophoresis is performed in short times As a rule, the        electrophoresis times to separate molecules up to 2 megabase        pairs are near to 8 hours.    -   5—The accessories, for casting gels and loading the sample plugs        into the wells, permit to cast gels of flat surfaces and to        align the loaded sample plugs lengthwise the migration origin.        This and the circulation of the solution at high flow velocity        with the attenuation of the turbulences of the fluid contribute        to obtain reproducible straight band patterns.    -   6—The system for pulling tight the electrodes avoids the        slackening of the electrodes, thus avoiding distortions of the        electric field force lines and, then, contributing to the        reproducibility of the band patterns.    -   7 —The system for pulling tight the electrodes facilitates that        the electrodes of TAFE chambers can be pulled tight by the        experimenter when they are slackened. The system for pulling        tight electrodes also has associated a system of elastic plugs        that seal the holes through which the electrodes are passed,        preventing the buffer solution from leaking even if the        electrode diameter reduces due to wear.    -   8—An accessory set for the preparation of thin sample plugs        having dimensions matching with the gel wells is provided.    -   9—The system used to place the electrodes in the chamber permits        to save platinum wire. As the chambers are small, other        materials used are also saved, lowering costs    -   10—A method is provided to determine the electrophoresis run        times for different experimental conditions. The method is based        on equations that describe the migration of DNA molecules in        CHEF chambers under pulsed field gel electrophoresis.    -   11—The gels used by minichambers are large enough to give        well-resolved band patterns., thus they are useful in analytical        and preparative applications. They are also wide enough to allow        to be loaded with numerous samples in a single experiment    -   12—The chambers support numerous UEZ, which can be activated or        occluded, as the experiment requires. Either type I or type II        TAFE chambers can have several UEZ, therefore they accommodate        one or several minigels and can analyze few or many samples. The        maximum number of samples ‘Nt’ that can be analyzed in an        electrophoresis is a multiple of the number of UEZ.    -   13—The co-electrophoresis of few or many samples is done in        short time. Either type I or type II TAFE chambers can separate        quickly the DNA molecules contained in several samples. For        example, when using four minigels, 80 seconds of pulse duration,        8 33 V/cm and 15° C., only 12 hours are required to separate the        Saccharomyces cerevisiae chromosomes.    -   14—The amount of reagents needed by type I and type II TAFE        chambers depends on the number of samples to be analyzed, and        thus, on the number of UEZ activated (‘Nzue’). It holds that the        volume of buffer filling the chamber is Bc=Nzue·Bnt.    -   15—Equations that permit to design optimally the dimensions of        TAFE chambers as well as to select the maximum values of the        electric field that can be applied in them are provided. The        maximum electric field that can be applied depends on the length        of the electrodes in type I TAFE chambers and on the number of        miniplatforms that will be activated in the type II TAFE        chambers, provided the rest of parameters of the equations are        kept constant and the power supply is properly selected.    -   16—Type I and type II TAFE chambers can use a little amount of        buffer solution because from them can be eliminated the NEZ,        regions crossed by electric field force lines that do not act on        the movement of molecules.    -   17—The chambers having the electrodes arranged in inverted TAFE        configuration are simple to construct and facilitate the        manipulation of minigels during the experiments    -   18—The minigels of type I and type II TAFE chambers use thin        samples, thus saving biological reagents and reducing the        electrophoresis run time.    -   19—Chambers with multiple UEZ are useful to perform molecular        epidemiology studies, to analyze strain collections, to analyze        YAC and BAC clones, and to perform any other application        involving a large number of samples

1. Pulsed field electrophoresis chambers with Transversal AlternatingField Electrophoresis (TAFE) electrode array having two pairs ofelectrodes, each pair formed by one cathode and one anode, forseparating DNA molecules immobilized in agarose plugs loaded into avertical gel by means of using a system for energizing the electrodesand alternating the direction of application of an electric fieldcrossing the gel transversally and generated by the electrode array, aswell as a system for circulating a buffer solution throughout thechamber, which TAFE chambers comprise: i) one or more minigels placed inzones crossed by lines of force of the electric field interactingdirectly with DNA molecules loaded into said minigel(s), wherein saidzones are Useful Electrophoresis Zones (UEZ) of said TAFE chambers, ii)said pairs of electrodes of opposite polarities, cathode and anode,separated in the TAFE electrode array a distance ‘d’, which is from 6.2to 15 cm; wherein said distance in conjunction with the number and sizesof said UEZs limit to certain values height, depth and width of saidTAFE chambers, length and width of said minigel(s), and total number ofsamples loaded simultaneously into all said minigel(s), iii) blocks ofmaterials of high dielectric constant occluding zones of said TAFEchambers crossed by the electric field force lines not acting on DNAmolecules loaded into said minigel(s), wherein said zones are non-usefulelectrophoresis zones (NEZ) zones of said TAFE chambers, iv) stretchedelectrodes, fixed and pulled tight by the action of a fixation and atension system, v) inverted TAFE electrode configuration in saidelectrode array, and vi) three accessory sets of said TAFE chambers forhomogenizing the electric current flowing through said chambers in theelectrophoresis; the first set comprising removable sheets that avoidturbulences of buffer circulated at high flow velocity; the second setcomprising disassemblable sets of frames, base plates, covers and combshaving teeth for casting said minigels with homogeneous cross sectionalarea and identical wells; and the third set comprising disassemblablesystems of grooved blocks or sample plug makers, covers of said groovedblocks, and cutters for preparing DNA samples immobilized in plugs forsaid minigels.
 2. Electrophoresis chambers of claim 1 wherein eachelectrode of said electrode array is parallel to a frontal wall of eachTAFE chamber and has a length (‘L’) of up to 50 cm wherein L is equal tothe width of said chamber.
 3. Electrophoresis chambers of claim 1wherein said minigels are d·0.515 cm in length and said length is from3.2 to 7.7 cm.
 4. Electrophoresis chambers of claim 1 wherein saidminigels are from 1.7 to 50 cm in width and have a number ‘NM’ of wellssupporting ‘NM’ number of DNA sample plugs, wherein said ‘NM’ is equalto (said minigel width in cm −0.2)/0.25.
 5. Electrophoresis chambers ofclaim 1 wherein said distance ‘d’ limits height and depth of said TAFEchambers by limiting to [2+1.4.·d]·[2+0.54·d]−1.02·[1+0.54·d]² cm² thearea of a chamber lateral wall, or wall supporting the electrodes andthe minigels, wherein said area is from 37.8 to 147.8 cm². 6.Electrophoresis chambers of claim 1 wherein said TAFE chambers areevenly subdivided into several UEZs with one minigel in each UEZ,wherein the number of UEZs is from 1 to
 30. 7. Electrophoresis chambersof claim 6 wherein said UEZs are as wide as each minigel and are fullyoccluded with rectangular blocks made of materials with high dielectricconstant.
 8. Electrophoresis chambers of claim 1 wherein said minigelsare arranged sequentially one next to the other, with their facesparallel to the electrodes.
 9. Electrophoresis chambers of claim 1wherein the electrode array of one of said TAFE chambers is in a fixedor removable single electrode platform (type I TAFE chamber), whereineach electrode of said electrode array is up to 50 cm in length.
 10. Theelectrophoresis chamber of claim 9 wherein said single electrodeplatform of said type I TAFE chamber is evenly subdivided and formsseveral UEZs with all minigels supported in a single frame.
 11. Theelectrophoresis chamber of claim 9 wherein said single electrodeplatform of said type I TAFE chamber is evenly subdivided and formsseveral UEZs with each minigel independently placed in each UEZ, whereinsaid minigels slide through laterally grooved pieces of said TAFEchambers.
 12. Electrophoresis chambers of claim 1 wherein saidelectrodes of one of said TAFE chambers are in various fixed orremovable independent mini-platforms, wherein each mini-platform limitsone useful electrophoresis zone (UEZ), has one minigel, and bears itselectrodes physically separated from the electrodes of the remainingmini-platforms (type II TAFE chamber); wherein said physically separatedelectrodes of one mini-platform acquire continuity with the electrodesof said remaining mini-platforms by plugging them in parallel, such thatsaid TAFE chamber is energized with a single power supply and samplesloaded into the minigels of the mini-platforms are exposed to the sameelectrophoresis conditions.
 13. Electrophoresis chambers of claim 1wherein said inverted TAFE electrode configuration comprises thecathodes of the electrode array being disposed at the bottom of theelectrophoresis chamber and the anodes at the top, so samples are loadedinto the minigel bottom and migrate in the direction opposite to thegravity.
 14. Electrophoresis chambers of claim 1 wherein one of saidblocks occluding NEZ of said TAFE chambers has its outer side parallelto the frontal wall of said TAFE chambers and its inner side forms asmall angle with a plane containing one of said cathodes and one of saidanodes located at the same side of the minigel(s).
 15. Electrophoresischambers of claim 1 wherein said stretched electrodes are fixed to saidTAFE chambers by the action of the fixation system, wherein said systemcomprises bored elastic plugs inserted into holes drilled into lateralwalls of said chambers, and said electrodes enter into said TAFEchambers from the outside passing through the bores of said elasticplugs.
 16. Electrophoresis chambers of claim 15 wherein said elasticplugs are made of silicone or rubber.
 17. Electrophoresis chambers ofclaim 1 wherein said system for pulling tight each electrode of saidTAFE chambers comprises i) a slotted rod with a hole crossing awaist-shaped notch, wherein said slotted rod bears the slot in its topside, ii) said electrode inserted into said hole, bent and wrappedaround said waist-shaped notch, iii) a grub screw for immobilizing orreleasing said slotted rod in desired position, wherein said slotted rodis located at the electrode exit of said TAFE chamber. 18.Electrophoresis chambers of claim 1 wherein said first set comprises tworemovable and identical sheets made of a material with high dielectricconstant, wherein each sheet is the size of the chamber wall parallel tothe electrodes and bears a horizontal slot in its lower third, whereinsaid slot is 0.3 cm in height and as long as each electrode. 19.Electrophoresis chambers of claim 18 wherein the two identical sheetsare placed as follows: one of said sheets near to the buffer inlet andthe other near to the outlet, and said sheets divide said TAFE chambersin three compartments: a central compartment containing the electrodesand the minigels, and two lateral compartments for delivering buffersolution into the chambers or withdrawing said buffer solution fromthem.
 20. Electrophoresis chambers of claim 1 wherein the second set ofsaid TAFE chambers comprises: i) a flat base plate, ii) two frames withtwo lateral notches for inserting the combs, wherein said frames arefrom 0.35 to 0.5 cm in thickness and have rectangular or square shapedcavities determining the shape, thickness (‘th’), length and width (‘a’)of each minigel cast in one of said frames, iii) a first comb, or combwith long teeth, iv) two covers: the first cover fitting against thefront of said first comb, and the second cover fitting against the backof said first comb, v) a second comb, similar to said first comb, butwith shorter teeth for pushing and aligning samples loaded into saidminigels.
 21. Electrophoresis chambers of claim 20 wherein said firstcomb is flat in its frontal part, and thicker over the teeth, forming astep in the rear, wherein said teeth are identical and from 0.03 to 0.1cm thick, from 0.15 cm to (a−0.3) cm in width, and (th−0.1) cm inlength.
 22. Electrophoresis chambers of claim 20 wherein said secondcomb has shape and sizes similar to said first comb, excepting thelength of the teeth, which are 0.2 cm shorter.
 23. Electrophoresischambers of claim 20 wherein the second cover has two flat surfaces anda protruding edge; and the first cover has two flat surfaces, but one ofits edges has a bevel cut in wedge formation.
 24. Electrophoresischambers of claim 1 wherein the third set of said TAFE chambers, forpreparing said DNA samples immobilized in plugs for minigels, comprises:i) various sample plug makers, wherein each sample plug maker is a flatimpermeable block thicker than 0.5 cm with multiple parallel grooveslengthwise, wherein each groove is 0.2 cm in width and from 0.03 to 0.1cm in depth, and said depth matches the thickness of teeth of a givencomb of said second set, ii) a flat rigid and impermeable sheet of atleast 0.1 cm in thickness, wich is a cover of said sample plug maker,iii) sample plug cutters, wherein each cutter is a bar as long as, orlonger than the grooves of one of said sample plug makers, and said barbears several protuberances with cutting edges in its inferior part andprotruding 0.1 cm from the bar, wherein said cutting edges are evenlyspaced, and said spacing is 0.15 cm at least.
 25. Electrophoresischambers of claim 1 wherein one of said TAFE chambers bearing a singleUEZ is energized at electric field strength up to 25 V/cm using a powersupply with maximum power output of 300 watt, and contains 0.5×TBEbuffer solution is used and maintained at constant temperature from 4 to30° C., wherein 1×TBE is 89 mM Tris, 89 mM boric acid and 2 mM EDTA, pH8.3.
 26. Electrophoresis chambers of claim 1 wherein one of said TAFEchambers with several UEZs is energized with a single power supply atelectric field strength from 8 to 25 V/cm,and contains 0.5×TBE buffersolution maintained at constant temperature from 4 to 30° C., wherein1×TBE is 89 mM Tris, 89 mM boric acid and 2 mM EDTA, pH 8.3.